担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1007/s00427-018-0623-x

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その他リンク： http://link.springer.com/content/pdf/10.1007/s00427-018-0623-x.pdf

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

In the amphibian intestine during metamorphosis, thyroid hormone (TH) induces some larval epithelial cells to dedifferentiate into stem cells, which generate the adult epithelium analogous to the mammalian intestinal epithelium. We have previously shown that the canonical Wnt signaling pathway is involved in adult epithelial development in the Xenopus laevis intestine. To understand the function of this pathway more precisely, we here focused on CD44, a major Wnt target, which has been identified as a TH response gene in the X. laevis intestine. Our in situ hybridization analysis indicated that CD44 mRNA is detectable in adult epithelial primordia consisting of the adult stem/progenitor cells and is strongly expressed in the connective tissue (CT) cells surrounding them. Interestingly, when the expression of CD44 mRNA is the highest, hyaluronan (HA), a principle ligand of CD44, is newly synthesized and becomes most abundantly distributed in the CT just beneath the adult epithelial primordia that are actively proliferating. Thereafter, as the adult primordia differentiate into the simple columnar epithelium, the expression of CD44 mRNA is gradually downregulated. More importantly, using organ cultures of the X. laevis tadpole intestine in the presence of TH, we have experimentally shown that inhibition of HA synthesis by 4-methylumbelliferone suppresses development of not only the CT but also the epithelial stem cells, resulting in failure to generate the AE. Our findings strongly suggest that TH-upregulated HA/CD44 signaling plays an essential role in formation of the intestinal stem cell niche during vertebrate postembryonic development.

DOI： 10.1002/stem.2671

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：NATURE PUBLISHING GROUP  

Thyroid hormone (T3) affects development and metabolism in vertebrates. We have been studying intestinal remodeling during T3-dependent Xenopus metamorphosis as a model for organ maturation and formation of adult organ-specific stem cells during vertebrate postembryonic development, a period characterized by high levels of plasma T3. T3 is believed to affect development by regulating target gene transcription through T3 receptors (TRs). While many T3 response genes have been identified in different animal species, few have been shown to be direct target genes in vivo, especially during development. Here we generated a set of genomic microarray chips covering about 8000 bp flanking the predicted transcription start sites in Xenopus tropicalis for genome wide identification of TR binding sites. By using the intestine of premetamorphic tadpoles treated with or without T3 and for chromatin immunoprecipitation assays with these chips, we determined the genome-wide binding of TR in the control and T3-treated tadpole intestine. We further validated TR binding in vivo and analyzed the regulation of selected genes. We thus identified 278 candidate direct TR target genes. We further provided evidence that these genes are regulated by T3 and likely involved in the T3-induced formation of adult intestinal stem cells during metamorphosis.

DOI： 10.1038/s41598-017-06679-x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ENDOCRINE SOC  

Most vertebrate organs use adult stem cells to maintain homeostasis and ensure proper repair when damaged. How such organ-specific stem cells are formed during vertebrate development is largely unexplored. We have been using the thyroid hormone (T3)-dependent amphibian metamorphosis to address this issue. Early studies in Xenopus laevis have shown that intestinal remodeling involves complete degeneration of the larval epithelium and de novo formation of adult stem cells through dedifferentiation of some larval epithelial cells. We have further discovered that the histidine ammonia-lyase (HAL; also known as histidase or histidinase)-2 gene is strongly and specifically activated by T3 in the proliferating adult stem cells of the intestine during metamorphosis, implicating a role of histidine catabolism in the development of adult intestinal stem cells. To determine the mechanism by which T3 regulates the HAL2 gene, we have carried out bioinformatics analysis and discovered a putative T3 response element (TRE) in the HAL2 gene. Importantly, we show that this TRE is bound by T3 receptor (TR) in the intestine during metamorphosis. The TRE is capable of binding to the heterodimer of TR and 9-cis retinoic acid receptor (RXR) in vitro and mediate transcriptional activation by liganded TR/RXR in frog oocytes. More importantly, the HAL2 promoter containing the TRE can drive T3-dependent reporter gene expression to mimic endogenous HAL2 expression in transgenic animals. Our results suggest that the TRE mediates the induction of HAL2 gene by T3 in the developing adult intestinal stem cells during metamorphosis.

DOI： 10.1210/en.2016-1558

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY  

In Xenopus laevis intestine during metamorphosis, the larval epithelial cells are removed by apoptosis, and the adult epithelial stem (AE) cells appear concomitantly. They proliferate and differentiate to form the adult epithelium (Ep). Thyroid hormone (TH) is well established to trigger this remodeling by regulating the expression of various genes including Notch receptor. To study the role of Notch signaling, we have analyzed the expression of its components, including the ligands (DLL and Jag), receptor ( Notch), and targets ( Hairy), in the metamorphosing intestine by real-time reverse transcription-polymerase chain reaction and in situ hybridization or immunohistochemistry. We show that they are up-regulated during both natural and TH-induced metamorphosis in a tissue-specific manner. Particularly, Hairy1 is specifically expressed in the AE cells. Moreover, upregulation of Hairy1 and Hairy2b by TH was prevented by treating tadpoles with a c-secretase inhibitor (GSI), which inhibits Notch signaling. More importantly, TH-induced up-regulation of LGR5, an adult intestinal stem cell marker, was suppressed by GSI treatment. Our results suggest that Notch signaling plays a role in stem cell development by regulating the expression of Hairy genes during intestinal remodeling. Furthermore, we show with organ culture experiments that prolonged exposure of tadpole intestine to TH plus GSI leads to hyperplasia of secretory cells and reduction of absorptive cells. Our findings here thus provide evidence for evolutionarily conserved role of Notch signaling in intestinal cell fate determination but more importantly reveal, for the first time, an important role of Notch pathway in the formation of adult intestinal stem cells during vertebrate development.

DOI： 10.1002/stem.2544

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1007/s00441-016-2396-8

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

Objective. Synovial fibroblasts (SFs) produce matrix-degrading enzymes that cause joint destruction in rheumatoid arthritis (RA). Epigenetic mechanisms play a pivotal role in autoimmune diseases. This study was undertaken to elucidate the epigenetic mechanism that regulates the transcription of matrix metalloproteinases (MMPs) in RASFs.
Methods. MMP gene expression and histone methylation profiles in the MMP promoters were examined in RASFs. The effect of WD repeat domain 5 (WDR5) silencing on histone methylation and MMP gene expression in RASFs was analyzed. MMP gene expression, surface expression of the interleukin-6 (IL-6) receptor, phosphorylation of STAT-3, and binding of STAT-3 in the MMP promoters were investigated in RASFs stimulated with IL-6.
Results. The MMP-1, MMP-3, MMP-9, and MMP-13 genes were actively transcribed in RASFs. Correspondingly, the level of histone H3 trimethylated at lysine 4 (H3K4me3) was elevated, whereas that of H3K27me3 was suppressed in the MMP promoters in RASFs. The decrease in H3K4me3 via WDR5 small interfering RNA reduced the levels of messenger RNA for MMP-1, MMP-3, MMP-9, and MMP-13 in RASFs. Interestingly, IL-6 signaling significantly increased the expression of MMP-1, MMP-3, and MMP-13, but not MMP-9, in RASFs. Although the IL-6 signaling pathway was similarly active in RASFs and osteoarthritis SFs, STAT-3 bound to the MMP-1, MMP-3, and MMP-13 promoters, but not the MMP-9 promoter, after IL-6 stimulation in RASFs.
Conclusion. Our findings indicate that histone methylation and STAT-3 regulate spontaneous and IL-6-induced MMP gene activation in RASFs. The combination of chromatin structure and transcription factors may regulate distinct arthritogenic properties of RASFs.

DOI： 10.1002/art.39563

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BIOMED CENTRAL LTD  

Background: RNA-binding protein Translocated in LipoSarcoma/FUsed Sarcoma (TLS/FUS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). We previously identified that TLS was associated with protein arginine methyltransferase 1 (PRMT1), and four arginine residues within TLS (R216, R218, R242 and R394) were consistently dimethylated. Protein arginine methylation is involved in various cellular events such as signal transduction, transcriptional regulation and protein-protein interactions.
Results: To understand the biological role of arginine methylation of RNA-binding protein, we prepared and characterized a mouse monoclonal antibody against asymmetric dimethylarginine of TLS. By cloning and screening, one stable hybridoma cell clone (2B12) producing anti-asymmetric dimethylated TLS on R216 and R218 antibody was established. The monoclonal antibody 2B12 is specific for the asymmetrically dimethylated arginine peptide and does not react with the same peptide sequence containing unmodified and symmetrically dimethylated arginine residues by dot-blot analysis. 2B12 was also validated GST tagged TLS with PRMT1 by in vitro arginine methylation assays. Since methylated TLS in HeLa cells and mouse and human brain protein extracts was immunoprecipitated with 2B12, we performed RNA-binding protein immunoprecipitation assays using HeLa cell lysate and this antibody. We demonstrated that the long noncoding RNA (lncRNA) transcribed from cyclin D1 promoter binds methylated TLS.
Conclusions: A monoclonal antibody that is capable of detecting the methylarginine status of TLS will facilitate the molecular and cellular analysis of transcriptional regulation by lncRNA through methylated TLS, and can be used as a favorable tool for clinical diagnosis of ALS caused by TLS dysregulation.

DOI： 10.1186/2045-3701-4-77

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

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. © 2013 Luu et al.
licensee BioMed Central Ltd.

DOI： 10.1186/2045-3701-3-43

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記述言語：英語   出版者・発行元：BIOMED CENTRAL LTD  

Thyroid hormone (T3) plays diverse roles in adult organ function and during vertebrate development. The most important stage of mammalian development affected by T3 is the perinatal period when plasma T3 level peaks. Amphibian metamorphosis resembles this mammalian postembryonic period and is absolutely dependent on T3. The ability to easily manipulate this process makes it an ideal model to study the molecular mechanisms governing T3 action during vertebrate development. T3 functions mostly by regulating gene expression through T3 receptors (TRs). Studies in vitro, in cell cultures and reconstituted frog oocyte transcription system have revealed that TRs can both activate and repress gene transcription in a T3-dependent manner and involve chromatin disruption and histone modifications. These changes are accompanied by the recruitment of diverse cofactor complexes. More recently, genetic studies in mouse and frog have provided strong evidence for a role of cofactor complexes in T3 signaling in vivo. Molecular studies on amphibian metamorphosis have also revealed that developmental gene regulation by T3 involves histone modifications and the disruption of chromatin structure at the target genes as evidenced by the loss of core histones, arguing that chromatin remodeling is an important mechanism for gene activation by liganded TR during vertebrate development.

DOI： 10.1186/2045-3701-2-42

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ENDOCRINE SOC  

Thyroid hormone (TH) plays critical roles during vertebrate postembryonic development. TH production in the thyroid involves incorporating inorganic iodide into thyroglobulin. The expression of iodotyrosine deiodinase (IYD; also known as iodotyrosine dehalogenase 1) in the thyroid gland ensures efficient recycling of iodine from the byproducts of TH biosynthesis: 3'-monoiodotyrosine and 3', 5'-diiodotyrosine. Interestingly, IYD is known to be expressed in other organs in adult mammals, suggesting iodine recycling outside the thyroid. On the other hand, the developmental role of iodine recycling has yet to be investigated. Here, using intestinal metamorphosis as a model, we discovered that the Xenopus tropicalis IYD gene is strongly up-regulated by TH during metamorphosis in the intestine but not the tail. We further demonstrated that this induction was one of the earliest events during intestinal metamorphosis, with IYD being activated directly through the binding of liganded TH receptors to a TH response element in the IYD promoter region. Because iodide is mainly taken up from the diet in the intestine and the tadpole stops feeding during metamorphosis when the intestine is being remodeled, our findings suggest that IYD transcription is activated by liganded TH receptors early during intestinal remodeling to ensure efficient iodine recycling at the climax of metamorphosis when highest levels of TH are needed for the proper transformations of different organs. (Endocrinology 153: 5082-5089, 2012)

DOI： 10.1210/en.2012-1308

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-BLACKWELL  

During amphibian metamorphosis, a series of dynamic changes occur in a predetermined order. Hind limb morphogenesis begins in response to low levels of thyroid hormone (TH) in early prometamorphosis, but tail muscle cell death is delayed until climax, when TH levels are high. It takes about 20 days for tadpoles to grow from early prometamorphosis to climax. To study the molecular basis of the timing of tissue-specific transformations, we introduced thyroid hormone receptor (TR) expression constructs into tail muscle cells of Xenopus tadpoles. The TR-transfected tail muscle cells died upon exposure to a low level of thyroxine (T4). This cell death was suggested to be mediated by type 2 iodothyronine deiodinase (D2) that converts T4 to T3the more active form of TH. D2 mRNA was induced in the TR-overexpressing cells by low levels of TH. D2 promoter contains a TH-response element (TRE) with a lower affinity for TR. These results show that the TR transfection confers the ability to respond to physiological concentrations of TH at early prometamorphosis to tail muscle cells through D2 activity and promotes TH signaling. We propose the positive feedback loop model to amplify the cell's ability to respond to low levels of T4.

DOI： 10.1111/j.1365-2443.2012.01614.x

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BIOMED CENTRAL LTD  

Background: Thyroid hormone (T3) is important for adult organ function and vertebrate development. Amphibian metamorphosis is totally dependent on T3 and offers a unique opportunity to study how T3 controls postembryonic development in vertebrates. Earlier studies have demonstrated that TR mediates the metamorphic effects of T3 in Xenopus laevis. Liganded TR recruits histone modifying coactivator complexes to target genes during metamorphosis. This leads to nucleosomal removal and histone modifications, including methylation of histone H3 lysine (K) 79, in the promoter regions, and the activation of T3-inducible genes.
Results: We show that Dot1L, the only histone methyltransferase capable of methylating H3K79, is directly regulated by TR via binding to a T3 response element in the promoter region during metamorphosis in Xenopus tropicalis, a highly related species of Xenopus laevis. We further show that Dot1L expression in both the intestine and tail correlates with the transformation of the organs.
Conclusions: Our findings suggest that TR activates Dot1L, which in turn participates in metamorphosis through a positive feedback to enhance H3K79 methylation and gene activation by liganded TR.

DOI： 10.1186/2045-3701-2-25

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担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Adult organ-specific stem cells are essential for organ homeostasis and tissue repair and regeneration. The formation of such stem cells during vertebrate development is poorly understood. Intestinal remodeling during thyroid hormone (T3)-dependent Xenopus metamorphosis resembles postembryonic intestinal maturation in mammals. During metamorphosis, the intestine is remodeled de novo via a yet unknown mechanism. Protein arginine methyltransferase 1 (PRMT1) is up-regulated in and required for adult intestinal stem cells during metamorphosis. PRMT1 up-regulation is the earliest known molecular event for the developing stem cells and is also conserved during zebrafish and mouse intestinal development. To analyze how PRMT1 is specifically up-regulated during the formation of the adult intestinal stem cells, wecloned the Xenopus PRMT1 promoter and characterized it in CaCo-2 cells, a human cell line with intestinal stem cell characteristics. Through a series deletion and mutational analyses, we showed that the stem cell-associated transcription factor c-Myc could bind to a conserved site in the first intron to activate the promoter. Furthermore, we demonstrated that during metamorphosis, both c-Myc and PRMT1 were highly up-regulated, specifically in the remodeling intestine but not the resorbing tail, and that c-Myc was induced by T3 prior to PRMT1 up-regulation. In addition, we showed that T3 directly activated the c-Myc gene during metamorphosis in the intestine via binding of the T3 receptor to the c-Myc promoter. These results suggest that T3 induces c-Myc transcription directly in the intestine, that c-Myc, in turn, activates PRMT1 expression, and that this is an important gene regulation cascade controlling intestinal stem cell development.

DOI： 10.1074/jbc.M111.335661

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ENDOCRINE SOC  

Thyroid hormone (T-3) plays an important role in regulating multiple cellular and metabolic processes, including cell proliferation, cell death, and energy metabolism, in vertebrates. Dysregulation of T-3 signaling results in developmental abnormalities, metabolic defects, and even cancer. We used T-3-dependent Xenopus metamorphosis as a model to study how T-3 regulates transcription during vertebrate development. T-3 exerts its metamorphic effects through T-3 receptors (TR). TR recruits, in a T-3-dependent manner, cofactor complexes that can carry out chromatin remodeling/histone modifications. Whether and how histone modifications change upon gene regulation by TR during vertebrate development is largely unknown. Here we analyzed histone modifications at T-3 target genes during intestinal metamorphosis, a process that involves essentially total apoptotic degeneration of the simple larval epithelium and de novo development of the adult epithelial stem cells, followed by their proliferation and differentiation into the complex adult epithelium. We demonstrated for the first time in vivo during vertebrate development that TR induces the removal of core histones at the promoter region and the recruitment of RNA polymerase. Furthermore, a number of histone activation and repression marks have been defined based on correlations with mRNA levels in cell cultures. Most but not all correlate with gene expression induced by liganded TR during development, suggesting that tissue and developmental context influences the roles of histone modifications in gene regulation. Our findings provide important mechanistic insights on how chromatin remodeling affects developmental gene regulation in vivo. (Endocrinology 153: 961-972, 2012)

DOI： 10.1210/en.2011-1736

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記述言語：英語   出版者・発行元：BIOMED CENTRAL LTD  

Adult organ-specific stem cells are essential for organ homeostasis and repair in adult vertebrates. The intestine is one of the best-studied organs in this regard. The intestinal epithelium undergoes constant self-renewal throughout adult life across vertebrates through the proliferation and subsequent differentiation of the adult stem cells. This self-renewal system is established late during development, around birth, in mammals when endogenous thyroid hormone (T3) levels are high. Amphibian metamorphosis resembles mammalian postembryonic development around birth and is totally dependent upon the presence of high levels of T3. During this process, the tadpole intestine, predominantly a monolayer of larval epithelial cells, undergoes drastic transformation. The larval epithelial cells undergo apoptosis and concurrently, adult epithelial stem/progenitor cells develop de novo, rapidly proliferate, and then differentiate to establish a trough-crest axis of the epithelial fold, resembling the crypt-villus axis in the adult mammalian intestine. We and others have studied the T3-dependent remodeling of the intestine in Xenopus laevis. Here we will highlight some of the recent findings on the origin of the adult intestinal stem cells. We will discuss observations suggesting that liganded T3 receptor (TR) regulates cell autonomous formation of adult intestinal progenitor cells and that T3 action in the connective tissue is important for the establishment of the stem cell niche. We will further review evidence suggesting similar T3-dependent formation of adult intestinal stem cells in other vertebrates.

DOI： 10.1186/2045-3701-1-30

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：PUBLIC LIBRARY SCIENCE  

Background: The intestinal epithelium undergoes constant self-renewal throughout adult life across vertebrates. This is accomplished through the proliferation and subsequent differentiation of the adult stem cells. This self-renewal system is established in the so-called postembryonic developmental period in mammals when endogenous thyroid hormone (T3) levels are high.
Methodology/Principal Findings: The T3-dependent metamorphosis in anurans like Xenopus laevis resembles the mammalian postembryonic development and offers a unique opportunity to study how the adult stem cells are developed. The tadpole intestine is predominantly a monolayer of larval epithelial cells. During metamorphosis, the larval epithelial cells undergo apoptosis and, concurrently, adult epithelial stem/progenitor cells develop de novo, rapidly proliferate, and then differentiate to establish a trough-crest axis of the epithelial fold, resembling the crypt-villus axis in the adult mammalian intestine. The leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a well-established stem cell marker in the adult mouse intestinal crypt. Here we have cloned and analyzed the spatiotemporal expression profile of LGR5 gene during frog metamorphosis. We show that the two duplicated LGR5 genes in Xenopus laevis and the LGR5 gene in Xenopus tropicalis are highly homologous to the LGR5 in other vertebrates. The expression of LGR5 is induced in the limb, tail, and intestine by T3 during metamorphosis. More importantly, LGR5 mRNA is localized to the developing adult epithelial stem cells of the intestine.
Conclusions/Significance: These results suggest that LGR5-expressing cells are the stem/progenitor cells of the adult intestine and that LGR5 plays a role in the development and/or maintenance of the adult intestinal stem cells during postembryonic development in vertebrates.

DOI： 10.1371/journal.pone.0013605

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Thyroid hormone (TH) affects diverse biological processes and can exert its effects through both gene regulation via binding the nuclear TH receptors (TRs) and non-genomic actions via binding to cell surface and cytoplasmic proteins. The critical importance of TH in vertebrate development has long been established, ranging from the formation of human cretins to the blockage of frog metamorphosis due the TH deficiency. How TH affects vertebrate development has been difficult to study in mammals due to the complications associated with the uterus-enclosed mammalian embryos. Anuran metamorphosis offers a unique opportunity to address such an issue. Using Xenopus as a model, we and others have shown that the expression of TRs and their heterodimerization partners RXRs (9-cis retinoic acid receptors) correlates temporally with metamorphosis in different organs in two highly related species, Xenopus laevis and Xenopus tropicalis. In vivo molecular studies have shown that TR and RXR are bound to the TH response elements (TREs) located in TH-inducible genes in developing tadpoles of both species. More importantly, transgenic studies in X. laevis have demonstrated that TR function is both necessary and sufficient for mediating the metamorphic effects of TH. Thus, the non-genomic effects of TH have little or no roles during metamorphosis and likely during vertebrate development in general. Published by Elsevier Inc.

DOI： 10.1016/j.ygcen.2010.01.019

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-LISS  

Matrix metalloproteinases (MMPs) play a pivotal role in development and/or pathogenesis through degrading extracellular matrix (ECM) components. We have previously shown that Xenopus MMP-9 gene is duplicated. To assess possible roles of MMP-9 and MMP-9TH in X. laevis intestinal remodeling, we here analyzed their expression profiles by in situ hybridization and show that their expression is transiently up-regulated during thyroid hormone-dependent metamorphosis. Of interest, MMP-9TH mRNA is strictly localized in the connective tissue and most highly expressed just beneath the larval epithelium that begins to undergo apoptosis. On the other hand, cells expressing MMP-9 mRNA become first detectable in the connective tissue and then, after the start of epithelial apoptosis, also in the larval epithelium. These results strongly suggest that MMP-9TH is responsible in the larval epithelial apoptosis through degrading ECM components in the basal lamina, whereas MMP-9 is involved in the removal of dying epithelial cells during amphibian intestinal remodeling.

DOI： 10.1002/dvdy.21252

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL PUBLISHING  

Several matrix metalloproteinases (MMP) are induced by thyroid hormone (TH) during the climax of amphibian metamorphosis and play a pivotal role in the remodeling of the intestine and the regressing tail and gills by degrading the extracellular matrix (ECM). We compared MMP gene expression levels precisely by quantitative real-time reverse transcription-polymerase chain reaction. The expression of MMP genes increases prominently at Nieuwkoop and Faber (NF) stages 60, 60-61 and 62 in the intestine, gills and tail, respectively, when the drastic morphological changes start in each organ. Gene expression analysis in the TH-treated tadpoles and cell line revealed that MMP mRNAs are upregulated in response to TH quickly within several hours to low levels and then increase in a day to high levels. All TH-induced MMP genes have TH response elements (TREs). The presence of high affinity TREs in MMP genes correlates with early TH-induction. Based on these results, we propose that TH stimulates the transcription of MMP genes through TREs within several hours to low levels and then brings about the main increase of mRNAs by TH-induced transcriptional factors, including TH receptor beta, in a cell type-specific transcriptional environment.

DOI： 10.1111/j.1440-169x.2007.00916.x

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：BLACKWELL PUBLISHING  

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.

DOI： 10.1111/j.1440-169x.2006.00859.x

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記述言語：英語   出版者・発行元：ACADEMIC PRESS LTD ELSEVIER SCIENCE LTD  

The death of different types of cells occurs in regressing or remodeling organs to transform from a tadpole to a frog in both temporally and spatially regulated manners during amphibian metamorphosis. This morphological change is drastic and visible with the naked eye. This review summarizes our current understanding of the basic mechanism of the cell death during the metamorphosis. It focuses in particular on the tail resorption and the remodeling of intestine and skin where programmed cell death is executed by thyroid hormone-signaling through the cell-autonomous response (suicide) and the degradation of the extracellular matrix (murder). (c) 2004 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.semcdb.2004.12.006

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-LISS  

In newt testis, spermatocytes never appear during winter, because secondary spermatogonia die by apoptosis just before meiosis. In the current study, we examined the effect of low temperatures on spermatogenesis. Incubation of newts at low temperatures (8, 12, 15degreesC) induced defects in spermatogenesis in a temperature-dependent manner. At 8degreesC, multinucleated giant cells (MGCs) were observed in spermatocytes and spermatogenesis never proceeded beyond meiosis. Although spermatocytes completed meiotic divisions at 12degreesC, severe cell death was observed in the spermatids. At 15degreesC both normal and abnormal spermiogenesis were observed. Under these conditions, impaired meiotic synapsis/recombination and down-regulation of the expression of the DMC1 protein, which play pivotal roles in meiotic pairing in eukaryotes, were also observed. Furthermore, to examine the quality of the sperm produced at low temperature for supporting development, artificial insemination was performed. The eggs inseminated with spermatozoa derived from newts kept at 15degreesC demonstrated a restricted developmental capacity, even though these spermatozoa had an equal capacity for carrying out fertilization to those kept at 22degreesC. These results suggest that meiosis at low temperatures cause the production of abnormal spermatozoa. Conservation and the significance of this phenomenon in poikilothermic vertebrates living in the temperate zones are also discussed. (C) 2003 Wiley-Liss, Inc.

DOI： 10.1002/mrd.10328

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ZOOLOGICAL SOC JAPAN  

We examined the effect of low temperatures on annexin V expression in newt testis. When newts were transferred to a low temperature (12degreesC), up-regulation of annexin V protein was observed in secondary spermatogonia. In primary spermatocytes, high levels of annexin V expression were observed at both 12degreesC and 22degreesC, but at 12degreesC the protein was localized in part of the cytoplasm of primary spermatocytes. These results indicate that in newt testis annexin V is a cold-sensitive protein, suggesting the possibility that annexin V might have a cold stress-related function in newt germ cells.

DOI： 10.2108/zsj.20.733

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ELSEVIER SCIENCE BV  

We previously showed that prolactin (PRL) induces apoptosis in newt secondary spermatogonia and indicated that caspase activity is involved in the apoptosis. Since it was recently reported that Z-VAD-fmk, a pan-caspase inhibitor, blocks activity of cysteine cathepsins as well, we examined whether cathepsin is involved in the newt spermatogonial apoptosis. We found cathepsin L activity in the testis that was elevated by PRL in organ culture of testis, while E-64d, a lysosomal cysteine protease inhibitor, and Z-VAD-fmk suppressed it and chromosomal condensation. These results suggest that cathepsin L activity play a pivotal role in PRL-induced spermatogonial apoptosis. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793%2802%2902817-X

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：ACADEMIC PRESS INC  

In newts elevated titers of plasma prolactin (PRL), induced by low temperature, cause apoptosis in the penultimate mitotic stage of spermatogonia, and this cell death is suppressed by antiserum against newt PRL, but only during the initial 3 days of exposure (Yazawa ct al., 1999). Thus, factors other than PRL must be involved in spermatogonial death. Follicle-stimulating hormone (FSH) may be a plausible candidate. Accordingly, the current study examined the activity of FSH on the proliferation and survival of spermatogonia at low temperatures in vivo and in vitro. Porcine FSH (pFSH) administration in vivo inhibited spermatogonial death induced at 12 degreesC, but failed to do so at 8 degreesC. Also pFSH promoted in vitro the proliferation of spermatogonia at 12 degreesC, but not at 8 degreesC. Furthermore, dibutyryl cyclic AMP stimulated in vitro DNA synthesis of secondary spermatogonia at 12 degreesC, but not at 8 degreesC. These different responses to temperatures were not caused by different levels of mRNA for the receptor of follicle-stimulating hormone, the number of FSH binding sites, or FSH binding affinity to its receptors in the testicular cells. Thus, the results indicate that a temperature-sensitive period exists during the postreceptor process and is responsible for the lack of response of newt testis to FSH at 8 degreesC. (C) 2001 Academic Press.

DOI： 10.1006/gcen.2001.7631

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：WILEY-LISS  

We previously showed in vivo and in vitro, that among the spermatogenic stages of the newt, prolactin (PRL) induces apoptosis specifically in the penultimate stage of, secondary spermatogonia. In the current report, we demonstrate in vitro that cycloheximide (CHX), an inhibitor of protein synthesis, induces morphological apoptotic changes similar to those caused by PRL, such as chromatin condensation and apoptotic body formation. Next, we found that Z-VAD-fmk, an inhibitor of various caspases, suppressed the apoptosis induced by PRL and CHX, but ICE inhibitor Ac-YVAD-CHO or caspase-3 inhibitor Ac-DEVD-CHO did not. As high caspase activity was present in extracts of testes treated with CHX, we suggest that an unidentified caspase induces the morphological changes of apoptosis in newt spermatogonia. Mol. Reprod. Dev. 59:209-214, 2001. (C) 2001 Wiley-Liss, Inc.

DOI： 10.1002/mrd.1024

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