Novel Rana keratin genes and their expression during larval to adult epidermal conversion in bullfrog tadpoles

The conversion of the larval to adult epidermis during metamorphosis of tadpoles of bullfrog, Rana catesbeiana, was investigated utilizing newly cloned Rana keratin cDNAs as probes. Rana larval keratin (RLK) cDNA (rlk) was cloned using highly specific antisera against Xenopus larval keratin (XLK). Tail skin proteins of bullfrog tadpoles were separated by 2-dimensional gel electrophoresis and subjected to Western blot analysis with anti-XLK antisera. The Rana antigen detected by this method was sequenced and identified as a type II keratin. We cloned rlk from tadpole skin by PCR utilizing primers designed from these peptide sequences of RLK. RLK predicted by nucleotide sequences of rlk was a 549 amino acid -long type II keratin. Subtractive cloning between the body and the tail skin of bullfrog tadpole yielded a cDNA (rak) of Rana adult keratin (RAK). RAK was a 433 amino acid-long type I keratin. We also cloned a Rana keratin 8 (RK8) cDNA (rk8) from bullfrog tadpole epidermis. RK8 was 502 amino acid-long and homologous to cytokeratin 8. Northern blot analyses and in situ hybridization experiments showed that rlk was actively expressed through prometamorphosis in larva-specific epidermal cells called skein cells and became completely inactive at the climax stage of metamorphosis and in the adult skin. RAK mRNA was expressed in basal cells of the tadpole epidermis and germinative cells in the adult epidermis. The expression of rlk and rak was down- and up-regulated by thyroid hormone (TH), respectively. In contrast, there was no change in the expression of RK8 during spontaneous and TH-induced metamorphosis. RK8 mRNA was exclusively expressed in apical cells of the larval epidermis. These patterns of keratin gene expression indicated that the expression of keratin genes is differently regulated by TH depending on the type of larval epidermal cells. The present study demonstrated the usefulness of these genes for the study of molecular mechanism of postembryonic epidermal development and differentiation.     

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.     

Amphibian organ remodeling during metamorphosis: insight into thyroid hormone-induced apoptosis

During amphibian metamorphosis, the animal body dramatically remodels to adapt from the aquatic to the terrestrial life. Cell death of larval organs/tissues occurs massively in balance with proliferation of adult organs/tissues, to ensure survival of the individuals. Thus, amphibian metamorphosis provides a unique and valuable opportunity to study regulatory mechanisms of cell death. The advantage of this animal model is the absolute dependence of amphibian metamorphosis on thyroid hormone (TH). Since the 1990s, a number of TH response genes have been identified in several organs of Xenopus laevis tadpoles such as the tail and the intestine by subtractive hybridization and more recently by cDNA microarrays. Their expression and functional analyses, which are still ongoing, have shed light on molecular mechanisms of TH‐induced cell death during amphibian metamorphosis. In this review, I survey the recent progress of research in this field, focusing on the X. laevis intestine where apoptotic process is well characterized at the cellular level and can be easily manipulated in vitro. A growing body of evidence indicates that apoptosis during the intestinal remodeling occurs not only via a cell‐autonomous pathway but also via cell–cell and/or cell–extracellular matrix (ECM) interactions. Especially, stromelysin‐3, a matrix metalloproteinase, has been shown to alter cell–ECM interactions by cleaving a laminin receptor and induce apoptosis in the larval intestinal epithelium. Here, I emphasize the importance of TH‐induced multiple apoptotic pathways for massive and well‐organized apoptosis in the amphibian organs and discuss their conservation in the mammalian organs.     

Apoptosis in amphibian organs during metamorphosis

During amphibian metamorphosis, the larval tissues/organs rapidly degenerate to adapt from the aquatic to the terrestrial life. At the cellular level, a large quantity of apoptosis occurs in a spatiotemporally-regulated fashion in different organs to ensure timely removal of larval organs/tissues and the development of adult ones for the survival of the individuals. Thus, amphibian metamorphosis provides us a good opportunity to understand the mechanisms regulating apoptosis. To investigate this process at the molecular level, a number of thyroid hormone (TH) response genes have been isolated from several organs of Xenopus laevis tadpoles and their expression and functional analyses are now in progress using modern molecular and genetic technologies. In this review, we will first summarize when and where apoptosis occurs in typical larva-specific and larval-to-adult remodeling amphibian organs to highlight that the timing of apoptosis is different in different tissues/organs, even though all are induced by the same circulating TH. Next, to discuss how TH spatiotemporally regulates the apoptosis, we will focus on apoptosis of the X. laevis small intestine, one of the best characterized remodeling organs. Functional studies of TH response genes using transgenic frogs and culture techniques have shown that apoptosis of larval epithelial cells can be induced by TH either cell-autonomously or indirectly through interactions with extracellular matrix (ECM) components of the underlying basal lamina. Here, we propose that multiple intra- and extracellular apoptotic pathways are coordinately controlled by TH to ensure massive but well-organized apoptosis, which is essential for the proper progression of amphibian metamorphosis.     

Characterization of Rana catesbeiana HSP30 gene and its expression in the liver of this amphibian during both spontaneous and thyroid hormone-induced metamorphosis

During metamorphosis, the Rana catesbeiana tadpole undergoes developmental changes in almost every tissue/organ. These changes prepare the ammonotelic, swimming larva for its transition to a ureotelic, terrestrial adult, and involve dramatic remodeling. The postembryonic changes in this tadpole are initiated by the thyroid hormones (TH) and result in the extensive degradation of proteins and degeneration of tissues characteristic of the larval phenotype and in the de novo synthesis of proteins characteristic of the adult phenotype. We questioned whether the drastic nature and abruptness of the TH-dependent, postembryonic changes occurring in the tissues of this tadpole might be perceived by the cells in some tissues as stressful and, therefore, cause them to express heat shock and/or stress-like proteins. To address this question, we isolated and characterized a Rana catesbeiana hsp30 gene and used sequences from it to determine if mRNAs encoded from it, or other members of this gene family, are expressed in tissues of tadpoles undergoing metamorphosis. Our results demonstrate that the liver of metamorphosing Rana catesbeiana tadpoles accumulate hsp30 mRNAs and express the heat shock proteins they encode. The fact that the expression of these hsp30s in the liver of these tadpoles is coincidental with the TH-induced expression of genes encoding the liver-specific urea cycle enzymes suggests that TH may influence, directly or indirectly, the expression of these hsp30 genes and, moreover, implies that the presence of one or more of these heat shock proteins may be necessary for the developmental transitions occurring in this organ. © 1996 Wiley-Liss, Inc.     

Erythropoiesis and unexpected expression pattern of globin genes in the salamander Hynobius retardatus

Transition of hemoglobin (Hb) from larval to adult types during the metamorphosis in a salamander Hynobius retardatus has been reported to occur almost independently of thyroid activity, in contrast to the case with many amphibians. In order to obtain further information on the mechanism of the transition in H. retardatus, larval and adult globin cDNAs were cloned, and the globin gene expression was analyzed in normally developing and metamorphosis-arrested animals. Northern hybridization and RT-PCR revealed that larval globin genes were initially expressed 5 days before hatching, and unexpectedly remained expressed even in juveniles 2 years old. The adult globin gene was expressed 19 days after hatching, much earlier than the initiation of morphological metamorphosis. Furthermore, the pattern of globin gene expression in metamorphosis-arrested larvae was almost identical to that in normal controls, suggesting that the transition occurs independently of thyroid hormones. In larvae recovering from anemia, precocious Hb transition, which occurs in Xenopus laevis and Rana catesbeiana, did not occur in H. retardatus. In situ hybridization convincingly demonstrated that the erythropoietic sites are the ventral blood island and the dorsolateral plate at the prehatching stage. During the ontogeny they changed to the liver, kidney, and spleen and were finally restricted to the spleen. Single erythroid cells expressed concurrently larval and adult globin genes, as demonstrated by double in situ hybridization. Thus the transition occurred within a single erythroid cell population, a unique characteristic of H. retardatus. Received: 5 August 1999 / Accepted: 14 October 1999     

Hormonal regulation of epidermis-specific protein and messenger RNA synthesis in amphibian metamorphosis

Experiments using a monospecific antibody directed against one type of epidermis-specific keratin from adult skin of the amphibian Xenopus laevis have demonstrated that polysomes synthesizing this protein first appear within larval skin during natural metamorphosis. Further experiments demonstrated that the synthesis of keratin within larval skin could be induced precociously by the thyroid hormone, 3,3′,5-triiodo-l-thyronine, both in vivo and when the isolated larval skin is cultured in vitro. The earliest developmental age responsive to such hormone induction appeared to be Stage $\text{50}\text{52}$ of larval development. This is about 20–24 days before keratin would normally make its appearance within the skin during natural metamorphosis. Hormone treatment of tadpoles at this age will also cause a precocious increase in the amount of keratin messenger RNA present within larval skin. This has been demonstrated directly by the isolation of poly(A)-containing messenger RNA from hormone-treated larvae and its translation in a wheat germ cell-free system to give immunoprecipitable keratin. Peptide analysis of the in vitro translation product indicates that the hormone-induced mRNA probably codes for an initial protein product that is slightly larger than keratin itself.     

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.     

One of the duplicated matrix metalloproteinase-9 genes is expressed in regressing tail during anuran metamorphosis

The drastic morphological changes of the tadpole are induced during the climax of anuran metamorphosis, when the concentration of endogenous thyroid hormone is maximal. The tadpole tail, which is twice as long as the body, shortens rapidly and disappears completely in several days. We isolated a cDNA clone, designated as Xl MMP-9TH, similar to the previously reported Xenopus laevis MMP-9 gene, and showed that their Xenopus tropicalis counterparts are located tandemly about 9 kb apart from each other in the genome. The Xenopus MMP-9TH gene was expressed in the regressing tail and gills and the remodeling intestine and central nervous system, and induced in thyroid hormone-treated tail-derived myoblastic cultured cells, while MMP-9 mRNA was detected in embryos. Three thyroid hormone response elements in the distal promoter and the first intron were involved in the upregulation of the Xl MMP-9TH gene by thyroid hormone in transient expression assays, and their relative positions are conserved between X. laevis and X. tropicalis promoters. These data strongly suggest that the MMP-9 gene was duplicated, and differentiated into two genes, one of which was specialized in a common ancestor of X. laevis and X. tropicalis to be expressed in degenerating and remodeling organs as a response to thyroid hormone during metamorphosis.     

Global gene expression profiling of Homarus americanus (Crustacea) larval stages during development and metamorphosis

Homarus americanus has a life history that is similar to other arthropods, including a pelagic larval phase and a benthic adult phase. The larval phase is divided into three morphologically distinct stages, followed by metamorphosis to the post-larval phase. H. americanus larval development has been studied previously, although the molecular mechanisms that regulate the consequent changes are not fully elucidated. This study is the first to use an oligonucleotide microarray to investigate global gene expression during H. americanus larval development. Stage-specific gene expression profiles of larvae and postlarvae from two-year classes were assessed. We found the expression levels of 1851 genes to be significantly different among larval stages. Functional annotations indicated that various differentially expressed genes were involved with immune function, energy regulation, and development. Ten target genes of interest were selected for expression verification using RT-qPCR. Two Phosphoenolpyruvatecarboxykinases, Argonaute 2, Ecdysone-inducible protein 75, and Procollagen-lysine 2-oxoglutarate 5-dioxygenase 3, had significantly different expression (p?相似文献   

Spatial and temporal expression pattern of a novel gene in the frog Xenopus laevis: correlations with adult intestinal epithelial differentiation during metamorphosis

We report the cloning of a novel gene (ID14) and its expression pattern in tadpoles and adults of Xenopus laevis. ID14 encodes a 315-amino acid protein that has a signal peptide and a nidogen domain. Even though several genes have a nidogen domain, ID14 is not the homolog of any known gene. ID14 is a late thyroid hormone (TH)-regulated gene in the tadpole intestine, and its expression in the intestine does not begin until the climax of metamorphosis, correlating with adult intestinal epithelial differentiation. In contrast, ID14 is expressed in tadpole skin and tail and is not regulated by TH. In situ hybridization revealed that this putative extracellular matrix protein is expressed in the epithelia of the tadpole skin and tail and in the intestinal epithelium after metamorphosis. In the adult, ID14 is found predominantly in the intestine with weak expression in the stomach, lung, and testis. Its exclusive expression in the adult intestinal epithelial cells makes it a useful marker for developmental studies and may give insights into cell/cell interactions in intestinal metamorphosis and adult intestinal stem cell maintenance.     

Differential regulation of two histidine ammonia-lyase genes during <Emphasis Type="Italic">Xenopus</Emphasis> development implicates distinct functions during thyroid hormone-induced formation of adult stem cells

Background

Organ-specific, adult stem cells are essential for organ-homeostasis and tissue repair and regeneration. The formation of such stem cells during vertebrate development remains to be investigated. Frog metamorphosis offers an excellent opportunity to study the formation of adult stem cells as this process involves essentially the transformations of all larval tissues/organs into the adult form. Of particular interest is the remodeling of the intestine. Early studies in Xenopus laevis have shown that this process involves complete degeneration of the larval epithelium and de novo formation of adult stem cells through dedifferentiation of some larval epithelial cells. A major advantage of this metamorphosis model is its total dependence on thyroid hormone (T3). In an effort to identify genes that are important for stem cell development, we have previously carried out tissue-specific microarray analysis of intestinal gene expression during Xenopus laevis metamorphosis.

Results

We report the detailed characterization of one of the genes thus identified, the histidine ammonia-lyase (HAL) gene, which encodes an enzyme known as histidase or histidinase. We show that there are two duplicated HAL genes, HAL1 and HAL2, in both Xenopus laevis and Xenopus tropicalis, a highly related but diploid species. Interestingly, only HAL2 is highly upregulated by T3 and appears to be specifically expressed in the adult intestinal progenitor/stem cells while HAL1 is not expressed in the intestine during metamorphosis. Furthermore, when analyzed in whole animals, HAL1 appears to be expressed only during embryogenesis but not metamorphosis while the opposite appears to be true for HAL2.

Conclusions

Our results suggest that the duplicated HAL genes have distinct functions with HAL2 likely involved in the formation and/or proliferation of the adult stem cells during metamorphosis.

     

Subtype-specific neuronal remodeling during Drosophila metamorphosis

During metamorphosis in holometabolous insects, the nervous system undergoes dramatic remodeling as it transitions from its larval to its adult form. Many neurons are generated through post-embryonic neurogenesis to have adult-specific roles, but perhaps more striking is the dramatic remodeling that occurs to transition neurons from functioning in the larval to the adult nervous system. These neurons exhibit a remarkable degree of plasticity during this transition; many subsets undergo programmed cell death, others remodel their axonal and dendritic arbors extensively, whereas others undergo trans-differentiation to alter their terminal differentiation gene expression profiles. Yet other neurons appear to be developmentally frozen in an immature state throughout larval life, to be awakened at metamorphosis by a process we term temporally-tuned differentiation. These multiple forms of remodeling arise from subtype-specific responses to a single metamorphic trigger, ecdysone. Here, we discuss recent progress in Drosophila melanogaster that is shedding light on how subtype-specific programs of neuronal remodeling are generated during metamorphosis.     

Gene switching at Xenopus laevis metamorphosis

During the climax of amphibian metamorphosis many tadpole organs remodel. The different remodeling strategies are controlled by thyroid hormone (TH). The liver, skin, and tail fibroblasts shut off tadpole genes and activate frog genes in the same cell without DNA replication. We refer to this as “gene switching”. In contrast, the exocrine pancreas and the intestinal epithelium dedifferentiate to a progenitor state and then redifferentiate to the adult cell type. Tadpole and adult globin are not present in the same cell. Switching from red cells containing tadpole-specific globin to those with frog globin in the liver occurs at a progenitor cell stage of development and is preceded by DNA replication. Red cell switching is the only one of these remodeling strategies that resembles a stem cell mechanism.     

Larval fat body cells die during the early pupal stage in the frame of metamorphosis remodelation in Bombyx mori

In holometabolus insects, morphology of the larval fat body is remodeled during metamorphosis. In higher Diptera, remodeling of the fat body is achieved by cell death of larval fat body cells and differentiation of the adult fat body from primordial cells. However, little is known about remodeling of the fat body at pupal metamorphosis in Lepidoptera. In this study, we found that cell death of the larval fat body in Bombyx mori occurs at shortly after pupation. About 30% of the fat body cells underwent cell death on days 1 and 2 after pupation. The cell death involved genomic DNA fragmentation, a characteristic of apoptosis. Surgical manipulation and in vitro culture of fat body cells revealed that 20-hydroxyecdysone and juvenile hormone had no effect on either initiation or progression of cell death. During cell death, a large increase in activity of caspase-3, a key enzyme of cell death, was observed. Western blot analysis of the active form of caspase-3-like protein revealed that the length of caspase-3 of B. mori was much larger than that of caspase-3 in other species. The results suggest that larval fat body cells of B. mori are removed through cell death, which is mediated by a caspase probably categorized in a novel family.