Molecular identification of the skin transformation center of anuran larval skin using genes of Rana adult keratin (RAK) and SPARC as probes

Anuran larval skin undergoes a process of metamorphosis into pre-adult and adult skin. Basal skein, larval basal and adult basal cells are basement membrane-attaching cells in the larval, pre-adult and adult epidermis, respectively, and are identified as cells expressing genes of RLK (Rana larval keratin), both RLK and RAK (Rana adult keratin), and RAK. Larval to pre-adult skin conversion takes place in the histological entity called the skin transformation center (STC). The present study performed a cDNA subtractive gene screening on cDNA of the larval and the pre-adult skin, and cloned the secreted protein acidic and rich in cysteine (SPARC) gene as an upregulated gene in the larva to pre-adult skin conversion. RAK gene-positive basal skein cells and fibroblasts in and around the STC were weakly and strongly sparc-positive, respectively. Using sparc and rak, we redefined the STC and visualized it on a histological section as an approximately 150 microm-long region that contained about 20 rak-negative and weakly sparc-positive basal cells. Intense sparc expression was observed in basal skein cells, but not in larval basal cells, suggesting that SPARC acts as a suppressor of rak during epidermal differentiation. This suggestion was tested by investigating the effect of SPARC on cultured larval basal cells. We observed that SPARC suppressed the expression of rak, but not rlk.     

Lineage of anuran epidermal basal cells and their differentiation potential in relation to metamorphic skin remodeling

The anuran remodels the larval epidermis into the adult one during metamorphosis. Larval and adult epidermal cells of the bullfrog were characterized by determining the presence of huge cytoplasmic keratin bundles and the expression profiles of specific marker genes, namely colalpha1 (collagen alpha1 (I)), rlk (larval keratin) and rak (adult keratin). We identified four types of epidermal basal cells: (i) basal skein cells that have keratin bundles and express colalpha1 and rlk; (ii) rak+-basal skein cells that have keratin bundles and express colalpha1, rlk, and rak; (iii) larval basal cells that express rlk and rak; and (iv) adult basal cells that express rak. These traits suggested that these basal cells are on the same lineage in which basal skein cells are the original progenitor cells that consecutively differentiate into rak+-basal skein cells into larval basal cells, and finally into adult basal cells. To directly verify the differentiation potential of larval basal cells into adult ones, the mono-layered epidermis composed of larval basal cells was cultured in the presence of aldosterone and thyroid hormone. In this culture, larval basal cells differentiated into adult basal cells that reconstituted the adult epidermis. Thus, it was concluded that larval basal cells are the direct progenitor cells of the adult epidermal stem cells.     

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.     

New epidermal keratin genes from Xenopus laevis: hormonal and regional regulation of their expression during anuran skin metamorphosis

Xenopus larval keratin (XLK) was isolated by gel electrophoresis of proteins of tadpole skin. Screening of an expression cDNA library of tail tissues by specific polyclonal antibodies against XLK produced XLK cDNA (xlk). Its complete nucleotide and predicted amino acid sequences revealed that XLK was a new member of type II keratin. Screening of a cDNA library of adult Xenopus skin using an oligonucleotide probe which had been designed from well-conserved N-terminal amino acid sequences of the rod domain of type I keratin produced two cDNAs, xak-a and xak-b, which were found to be new members of type I keratin gene. Northern blot analysis showed that xlk was expressed exclusively in the larval skin whereas xak-a and xak-b were expressed exclusively in the adult skin. Their expression level was regulated in a region- and metamorphic stage- dependent manner during larval skin development. mRNA in situ hybridization experiments identified the cells that expressed xlk, and xak-a and xak-b as larva- specific epidermal cells (skein cells and basal cells), and adult suprabasal epidermal cells, respectively. These three genes were found to be late responsive to thyroid hormone. Phylogenetic relationships of these keratins with known ones are discussed.     

Characterization of metamorphic changes in anuran larval epidermis using lectins as probes

The skin of an adult frog of Xenopus laevis was characterized by the reactivity of 20 lectins. The lectins were classified into six groups in their binding to the epidermal cells: Lycopersicon esculentum lectin (LEL)-type which was positive for all epidermal cells; Pisum sativum agglutinin (PSA)-type for stratum germinativum; succinylated wheat germ agglutinin (sWGA)-type for strata spinosum, granulosum and corneum; Dolichos biflorus agglutinin (DBA)-type for strata germinativum and spinosum; peanut agglutinin (PNA)-type for stratum spinosum; and Ulex europaeus agglutinin (UEA-I)-type for strata granulosum and corneum. PSA and sWGA were utilized as markers of mitotically active germinative cells and the differentiated cells of the epidermis, respectively, to describe the metamorphic conversion of larval epidermal cells to adult type. PSA stained all epidermal cells of tadpoles before metamorphic climax. At the end of metamorphosis, PSA-positive cells were restricted to cells in the basal layer of body epidermis while all the tail epidermis remained PSA-positive. The other cell marker, sWGA, only stained apical cells in tadpole epidermis. During the metamorphic climax, sWGA-positive cells appeared in the cells beneath the stratum corneum of the body region, but not in the tail region. The present study demonstrates that PSA and sWGA are useful to investigate metamorphic changes in tadpole epidermal cells.     

In Vitro Regression of Tadpole Tail by Thyroid Hormone

DERBY successfully maintained the tail of tadpole ( Rana pipiens) in vitro over a period of 2 weeks in a physiological salt solution (1). When we tried to apply DERBY'S methods of the tissue culture to tadpoles of bullfrog, Rana catesbeiana , it was found that the tissue regressed spontanously without stimulation of thyroid hormone. Several different media were examined in order to select a better culture medium for the bullfrog tadpole tissues. RPMI-1640 medium supplemented with insulin and transferrin was found to be satisfactory for this aim. With this improved medium, the interaction between the epidermis and the mesenchyme has been investigated during the hormone-induced tadpole tail regression and the epidermal dependence of the mesenchyme regression was demonstrated by the following three experiments. (i) Some of surgically prepared mesenchymes regressed in responce to thyroid hormone. In these cases the mesenchymes were revealed to be contaminated with the remaining epidermal cells. (ii) Complete removal of the epidermis was accomplished by the chemical treatment. The mesenchyme thus obtained ("nude tail fin") was insensible to thyroid hormone. (iii) "Skin conditioned medium" (SCM) was prepared by culturing the skin in the presence and absence of thyroid hormone. Nude tail fin regressed when cultured in the SCM containing thyroid hormone.     

Molecular,cellular and histological changes in skin from a larval to an adult phenotype during bony fish metamorphosis

Developmental models for skin exist in terrestrial and amphibious vertebrates but there is a lack of information in aquatic vertebrates. We have analysed skin epidermal development of a bony fish (teleost), the most successful group of extant vertebrates. A specific epidermal type I keratin cDNA (hhKer1), which may be a bony-fish-specific adaptation associated with the divergence of skin development (scale formation) compared with other vertebrates, has been cloned and characterized. The expression of hhKer1 and collagen 1α1 in skin taken together with the presence or absence of keratin bundle-like structures have made it possible to distinguish between larval and adult epidermal cells during skin development. The use of a flatfish with a well-defined larval to juvenile transition as a model of skin development has revealed that epidermal larval basal cells differentiate directly to epidermal adult basal cells at the climax of metamorphosis. Moreover, hhKer1 expression is downregulated at the climax of metamorphosis and is inversely correlated with increasing thyroxin levels. We suggest that, whereas early mechanisms of skin development between aquatic and terrestrial vertebrates are conserved, later mechanisms diverge. This work was carried out within the project “Arrested development: The Molecular and Endocrine Basis of Flatfish Metamorphosis” (Q5RS-2002-01192) with financial support from the Commission of the European Communities. It does not necessarily reflect the Commission’s views and in no way anticipates its future policy in this area. This project was further supported by Pluriannual funding to CCMAR by the Portuguese Science and Technology Council. M.A. Campinho was sponsored by the Portuguese Ministry of Science (grant no. SFRH/BD/6133/2001).     

Developmentally and regionally regulated participation of epidermal cells in the formation of collagen lamella of anuran tadpole skin

We investigated the cellular mechanism of formation of subepidermal thick bundles of collagen (collagen lamella) during larval development of the bullfrog, Rana catesbeiana, using cDNA of alpha1(I) collagen as a probe. The originally bilayered larval epidermis contains basal skein cells and apical cells, and the collagen lamella is directly attached to the basement membrane. The basal skein cells above the collagen lamella and fibroblasts beneath it intensively expressed the alpha1(I) gene. As the skin developed, suprabasal skein cells ceased expression of the gene. Concomitantly, the fibroblasts started to outwardly migrate, penetrated into the lamella and formed connective tissue between the epidermis and the lamella. These fibroblasts intensively expressed the gene. As the connective tissue developed, the basal skein cells ceased to express the gene and were replaced by larval basal cells that did not express the gene. These dynamic changes took place first in a lateral region of the body skin and proceeded to all other regions except the tail. Isolated cultured skein cells expressed the gene and extracellularly deposited its protein as the type I collagen fibrils. Thus, it is concluded that anuran larval epidermal cells can autonomously and intrinsically synthesize type I collagen.     

Adult precursor cells in the tail epidermis of Xenopus tadpoles

Polyclonal antibodies were raised against Xenopus larva-specific 58 kDa keratin (PAK58) and adult-specific 63 kDa keratin (PAK63), in order to examine the origin of 63 kDa-keratin-producing cells in the tail skin. By immunofluorescent staining of the tail skin, the 58 kDa keratin was recognized in almost all of the larval epidermal cells, although a small number of PAK58-negative cells were detected at stage 64. In contrast, 63 kDa keratin was immunohistochemically recognized at stage 58, but the signal was very weak. The number of epidermal layers in the tail epidermis increased during a period from stage 58 to stage 64. At stage 64, a small number of PAK63-positive cells was clearly identified in the multilayered tail epidermis. Comparative analysis of successive sections showed that PAK63-positive cells are derived from a cell group differing from PAK58-positive cells. Immunohistochemical studies using cultured epidermal cells demonstrated that 58 kDa keratin is localized in the cytoskeletal bundles of skein cells, whereas 63 kDa keratin is produced not by skein cells but by basal cells and their descendants. These results suggest that basal cells are the adult precursor cells within the larval epidermis even in the tail area.     

Biochemical and Immunological Characterization of Collagenase in Tissues of Metamorphosing Bullfrog Tadpoles

Tissue inhibitor of metalloproteinases (TIMP, a specific inhibitor of collagenase) was found to inhibit thyroid hormone-induced tail regression, suggesting the important role of collagenase in this process. Collagenase was purified from culture media of back skin of tadpole of bullfrog, Rana catesbeiana . Anti-tadpole collagenase polyclonal antisera were obtained against the purified enzyme. The antibody inhibited the activity of tadpole collagenase. The antisera reacted to tissues of adult bullfrogs, tadpoles of african clawed frog, Xenopus laevis , and adult newts, Cynopus pyrrhogaster , and also reacted to human fibroblast collagenase. Immunoblot analyses suggested that tadpole collagenase lacks the procollagenase which is generally found in mammalian collagenases. Intense immunological stains were observed for the tissues of thyroid hormone-treated tadpoles as compared to those of untreated animals. Thyroid hormone increased amounts of collagenase not only in epidermal layer but also in mesenchymal tissues including fibroblastic cells.     

Visualization of the initiation and sequential expansion of the metamorphic conversion of anuran larval skin into the precursor of adult type

A tadpole of bullfrog, Rana catesbeiana , is originally covered with the larval skin over its entire body. Drastic changes arise in both the epidermis and the subcutaneous connective tissue at an early developmental stage, producing the precursor of adult type skin (pre-adult skin). It was found that calcium is a useful probe to detect the region where the precursor formation has occurred because its deposition in the upper part of subcutaneous collagen bundles coincides with the appearance of the pre-adult skin. Whole-mount in situ staining of tadpoles with alizarin red S revealed the initiation site of the premetamorphic transformation of the larval skin into the adult precursor and its ensuing region-dependent expansion. The pre-adult skin first emerged at TK II to III (TK, Taylor and Kollros staging) t lateral sides of the body, which led us to postulate that 'the center for premetamorphic skin transformation' is formed at the specific site in this region. This center moved dorsally and then ventrally, then reached to the most proximal region of the tail, yielding a unique sequential conversion pattern by around TK V when the conversion was completed in the trunk. The present study also visualized the process of the hindlimb skin transformation.     

Hormonal regulation of adult type keratin gene expression in larval epidermal cells of the frog Xenopus laevis

Triiodothyronin (T3) is known to induce amphibian metamorphosis but other hormones such as glucocorticoids accelerate T3 action. The increase in plasma concentration of both T3 and glucocorticoids during metamorphic climax is correlated with the transformation of the epidermis from larval type (uncornified) to adult type (cornified). Previously we have shown that T3 induced adult-type 63 Kd keratin gene expression and cornification of the larval epidermis. In this study, we have examined the effects of T3 and hydrocortisone (HC) on the conversion of larval to adult epidermal cells in vitro. When larval epidermal cells were treated with both T3 and HC, they had a synergistic effect on adult-type keratin synthesis (both 63 Kd and 49 Kd keratins) and epidermal cornification. The synergistic effect between T3 and HC required a pretreatment with T3 for 3 days. During this time, addition of HC to cultures containing T3 did not change the amount of 63 Kd keratin mRNA. Thus, HC did not reduce the lag time for epidermal cells to respond to T3. After 4 days of hormone treatment, T3 increased the amount of 63 Kd keratin mRNA 9-fold while T3 and HC induced it 18-fold. When cultures were pretreated with T3 for 3 days, a 1 day treatment with HC was sufficient to obtain the synergistic effect. Thus the induction of 63 Kd keratin gene expression by T3 required a much longer lag (3 days) than the lag required for the synergistic action of T3 and HC (less than 1 day).(ABSTRACT TRUNCATED AT 250 WORDS)     

The epidermis and its degeneration in the larval tail and adult body of Rana temporaria and Xenopus laevis (Amphibia: Anura)

The tail epidermis of the larva and the body epidermis of adults of Rana temporaria and Xenopus laevis are described in terms of electron microscopy.
The activity of lysosomes (determined by the localization of acid phosphatase) in relation to autolysis and the process of cellular cornification, is considered during the periods of climactic disappearance of the larval tail and skin sloughing of adults. The results obtained generally correspond for both genera.
Larval tail epidermal cells completely disappear at metamorphic climax; those of the adult, which are shed, are replaced throughout life after each periodic sloughing. Nevertheless the mechanisms of their epidermal cell loss are comparable, though the level of lysosomal activity in larval tail epidermal cells is higher than in the adult body epidermis. This higher activity of lysosomal enzymes may facilitate the heavy necrosis which ensues in the larval tail at metamorphic climax.     

Spatial, temporal, and hormonal regulation of epidermal keratin expression during development of the frog, Xenopus laevis.

To study the mechanism of hormone-induced keratin expression in the epidermis during Xenopus metamorphosis, a monospecific antibody was raised against a unique carboxy-terminal peptide of the 63-kDa keratin. Immunohistological analysis demonstrated that the onset of 63-kDa keratin expression showed distinct regional and temporal differences. The expression started at stage 54 in the hindlimb epidermis, at stage 57 in the head, and over 1 month later at stage 63 in the tail. The amount of 63-kDa keratin was further regulated during epidermal stratification and differentiation. The 63-kDa keratin was expressed first in basal epidermal cells before stratification began. The outer layer of the larval epidermis (periderm) did not express the 63-kDa keratin. As the cells moved out of basal layer, they stained more intensely with the anti-keratin antibody indicating that 63-kDa keratin synthesis is up-regulated during differentiation. Similar results were obtained with cultures of purified epidermal cells grown in high calcium conditions. Since we have shown that thyroid hormone (T3) induces 63-kDa keratin gene expression and hydrocortisone (HC) modulates T3 action we examined the effects of T3 and HC at the single cell level with the anti-keratin antibody. Immunostaining demonstrated that T3 alone and T3 plus HC increased the number of 63-kDa keratin-positive cells as well as the amount of 63-kDa keratin per cell. Unexpectedly these hormones had the same effects on head and tail epidermal cells even though the latter cells degenerate during metamorphosis. The major difference between tail and head cells was that the percentage 63-kDa keratin-producing cells was much greater in the head than in the tail.     

Spatial and temporal expression patterns of Xenopus Nkx-2.3 gene in skin epidermis during metamorphosis

Using PCR cloning, the mRNA of XNkx-2.3 gene, a Xenopus tinman homologue, was identified in a cDNA library prepared from thyroid hormone (T(3))-treated tadpole skin. Quantitative RT-PCR and RNase Protection Assay confirmed the expression of XNkx-2.3 in adult frog skin and its amount was similar to the amount found in heart. In situ hybridization indicated that XNkx-2.3 was expressed in the frog epidermis. Further analysis of XNkx-2.3 expression patterns demonstrates that it shares great similarities with a 63 kDa keratin, a well-characterized marker for skin maturation, in the following aspects. First, XNkx-2.3 was expressed in tadpole skin during metamorphosis (stages 55-59), but not in pre-metamorphic (stage 54) skin. Secondly, XNkx-2.3 expression in skin responded to T(3) stimulation because it could be precociously induced by T(3) at pre-metamorphic stage, both in tadpoles and in cultures of skin explants. Finally, the T(3)-induced appearance of XNkx-2.3 in head skin occurred earlier and at higher level than that in tail skin. These data suggest that XNkx-2.3 may be an important factor for skin maturation and may also serve as a good marker to indicate the maturation of Xenopus 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.     

Loss of reactivity to pan-cadherin antibody in epidermal cells as a marker for metamorphic alteration of Xenopus skin

Pan-cadherin antibodies recognize the conserved C-terminal region of the family of cell-cell adhesion molecules, cadherins, and have a broad spectrum of reactivity to the molecules. In the present study, by immunohistochemistry using an anti-pan cadherin monoclonal antibody (mAb), expression dynamics of cadherins in epidermal tissues were analyzed during metamorphosis of Xenopus laevis. At early stages of development, the anti-pan cadherin mAb detected signals at cell-cell boundaries and in the cytoplasm of both trunk and tail epidermal cells. During metamorphosis, the immunoreactivity decreased in the trunk skin tissue but remained in the tail. At the climax stage, immunoreactivity was observed only in the regressing tail epidermis. The signals disappeared completely from the trunk epidermis, which had already transformed into adult-type tissue. This observation was confirmed by western blot analysis. A specific band was detected in the larval skin, but not in the adult lysate, at approximately 135 kDa in molecular size, corresponding to the molecular mass of cadherins. This different immunoreactivity in larvae and adults was observed in the epidermis of the skin, but not in any other tissues examined, that is, brain, kidney and liver. The immunoreactivity seen in larval epidermal cells was drastically downregulated by thyroid hormone treatment in vitro. These changes of immunoreactivity were specific for the C-terminal region of cadherins, suggesting intracellular alteration of the molecules during metamorphosis, and the anti-pan cadherin mAb can be a marker for larval-type epidermal cells that is applicable to analysis of Xenopus metamorphosis.     

Larval antigen molecules recognized by adult immune cells of inbred Xenopus laevis: partial characterization and implication in metamorphosis

It has been shown that larval skin (LS) grafts are rejected by an inbred strain of adult Xenopus, which suggests a mechanism of metamorphosis by which larval cells are recognized and attacked by the newly differentiating immune system, including T lymphocytes. In an attempt to define the larval antigenic molecules that are targeted by the adult immune system, anti-LS antibodies (IgY) were produced by immunizing adult frogs with syngeneic LS grafts. The antigen molecules that reacted specifically with this anti-LS antiserum were localized only in the larval epidermal cells. Of 53 and 59-60 kDa acidic proteins that were reactive with anti-LS antibodies, a protein of 59 kDa and with an isoelectric point of 4.5 was selected for determination of a 19 amino acid sequence (larval peptide). The rat antiserum raised against this peptide was specifically reactive with the 59 kDa molecules of LS lysates. Immunofluorescence studies using these antisera revealed that the larval-specific molecules were localized in both the tail and trunk epidermis of premetamorphic larvae, but were reduced in the trunk regions during metamorphosis, and at the climax stage of metamorphosis were detected only in the regressing tail epidermis. Culture of splenocytes from LS-immunized adult frogs in the presence of larval peptide induced augmented proliferative responses. Cultures of larval tail pieces in T cell-enriched splenocytes from normal frogs or in natural killer (NK)-cell-enriched splenocytes from early thymectomized frogs both resulted in significant destruction of tail pieces. Tissue destruction in the latter was enhanced when anti-LS antiserum was added to the culture. These results indicate that degeneration of tail tissues during metamorphosis is induced by a mechanism such that the larval-specific antigen molecules expressed in the tail epidermis are recognized as foreign by the newly developing adult immune system, and destroyed by cytotoxic T lymphocytes and/or NK cells.