Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

During frog gastrulation, mesendodermal cells become apposed to the blastocoel roof (BCR) by endoderm rotation, and migrate towards the animal pole. The leading edge of the mesendodermal cells (LEM) contributes to the directional migration of involuting marginal zone (IMZ) cells, but the molecular mechanism of this process is not well understood. Here we show that CXCR4/SDF-1 signaling mediates the directional movement of the LEM in Xenopus embryos. Expression of xCXCR4 was detected in the IMZ, and was complemented by xSDF-1 alpha expression in the inner surface of the BCR. Over-expression of xCXCR4 and xSDF-1 alpha caused gastrulation defects. An xCXCR4 N-terminus deletion construct and xSDF-l alpha-MO also inhibited gastrulation. Furthermore, explants of LEM migrate towards the dorsal BCR in the presence of xSDF-1a, and altered xCXCR4 expression in the LEM inhibited LEM migration. These results suggest that CXCR4/SDF-1 signaling is necessary for the migrations of massive numbers of cells during gastrulation. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.01.007

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

There are several lines of evidence that activin is a crucial molecule for mesoderm induction in early Xenopus embryos. However, it is not known what kind and what amounts of activin proteins exist in cleavage stage embryos. Three isc,forms of activins, A, AB, and B, were demonstrated to be present at least in part as a complex with abundant follistatin, an activin-binding protein, in early Xenopus embryos (stage 1-5). These results suggest that activin stored in eggs has an important role for mesoderm induction during early Xenopus embryogenesis and that follistatin can modulate the function of activin in signaling developmental changes. (C) 1994 Academic Press, Inc.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Genetic sex-determining systems in vertebrates include two basic types of heterogamety; XX (female)/ XY (male) and ZZ (male)/ZW (female) types. The African clawed frog Xenopus laevis has a ZZ/ZW-type sex-determining system. In this species, we previously identified a W-specific sex (female)-determining gene dmw, and specified W and Z chromosomes, which could be morphologically indistinguishable (homomorphic). In addition to dmw, we most recently discovered two genes, named scanw and ccdc69w, and one gene, named capn5z in the W-and Z-specific regions, respectively. In this study, we revealed the detail structures of the W/Z-specific loci and genes. Sequence analysis indicated that there is almost no sequence similarity between 278 kb W-specific and 83 kb Z-specific sequences on chromosome 2Lq3233, where both the transposable elements are abundant. Synteny and phylogenic analyses indicated that all the W/Z-specific genes might have emerged independently. Expression analysis demonstrated that scanw and ccdc69w or capn5z are expressed in early differentiating ZW gonads or testes, thereby suggesting possible roles in female or male development, respectively. Importantly, the sex-determining gene (SDG) dmw might have been generated after allotetraploidization, thereby indicating the construction of the new sex-determining system by dmw after species hybridization. Furthermore, by direct genotyping, we confirmed that diploid WW embryos developed into normal female frogs, which indicate that the Z-specific region is not essential for female development. Overall, these findings indicate that sex chromosome differentiation has started, although no heteromorphic sex chromosomes are evident yet, in X laevis. Homologous recombination suppression might have promoted the accumulation of mutations and transposable elements, and enlarged the W/Z-specific regions, thereby resulting in differentiation of the W/Z chromosomes. (C) 2016 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2016.06.015

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Xenopus laevis has an allotetraploid genome of 3.1 Gb, in contrast to the diploid genome of a closely related species, Xenopus tropicalis. Here, we identified 412 genes (189 homeolog pairs, one homeologous gene cluster pair, and 28 singletons) encoding transcription factors (TFs) in the X. laevis genome by comparing them with their orthologs from X. tropicalis. Those genes include the homeobox gene family (Mix/Bix, Lhx, Nkx, Paired, POU, and Vent), Sox, Fox, Pax, Dmrt, Hes, GATA, T-box, and some clock genes. Most homeolog pairs for TFs are retained in two X. laevis subgenomes, named L and S, at higher than average rates (87.1% vs 60.2%). Among the 28 singletons, 82.1% were deleted from chromosomes of the S subgenome, a rate similar to the genome-wide average (82.1% vs 74.6%). Interestingly, nkx2-1, nkx2-8, and pax9, which reside consecutively in a postulated functional gene cluster, were deleted from the S chromosome, suggesting cluster-level gene regulation. Transcriptome correlation analysis demonstrated that TF homeolog pairs tend to have more conservative developmental expression profiles than most other types of genes. In some cases, however, either of the homeologs may show strongly different spatio-temporal expression patterns, suggesting neofunctionalization, subfunctionalization, or nonfunctionalization after allotetraploidization. Analyses of otxl suggests that homeologs with much lower expression levels have undergone greater amino acid sequence diversification. Our comprehensive study implies that TF homeologs are highly conservative after allotetraploidization, possibly because the DNA sequences that they bind were also duplicated, but in some cases, they differed in expression levels or became singletons due to dosage-sensitive regulation of their target genes.

DOI： 10.1016/j.ydbio.2016.09.017

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Language：English   Publisher：(公社)日本生化学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL  

Dynamic changes of cytoplasmic and cortical actin filaments drive various cellular and developmental processes. Although real-time imaging of actin filaments in living cells has been developed, imaging of actin filaments in specific cells of living organisms remains limited, particularly for the analysis of gamete formation and early embryonic development. Here, we report the production of transgenic zebrafish expressing the C-terminus of Moesin, an actin filament-binding protein, fused with green fluorescent protein or red fluorescent protein (GFP/RFP-MoeC), under the control of a cyclin B1 promoter. GFP/RFP-MoeC was expressed maternally, which labels the cortical actin cytoskeleton of blastula-stage cells. High levels of GFP/RFP fluorescence were detected in the adult ovary and testis. In the ovaries, GFP/RFP-MoeC was expressed in oocytes but not in follicle cells, which allows us to clearly visualize the organization of actin filaments in different stages of the oocyte. Using full-grown oocytes, we revealed the dynamic changes of actin columns assembled in the cortical cytoplasm during oocyte maturation. The number of columns slightly decreased in the early period before germinal vesicle breakdown (GVBD) and then significantly decreased at GVBD, followed by recovery after GVBD. Our transgenic fish are useful for analyzing the dynamics of actin filaments in oogenesis and early embryogenesis. (C) 2015 Wiley Periodicals, Inc.

DOI： 10.1002/cm.21253

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Introduction of epithelial lumen-like structures such as blood and lymphatic vessels, as well as renal tubules, is a prerequisite for successful construction and function of artificially engineered giant tissues. Here, we demonstrate a methodology for construction of various epithelial lumen-like structures by using multichannel collagen gels (MCCGs). MCCGs were prepared and used as template scaffolds for constructing epithelial lumen structures in a controlled fashion. The effect of NaCl concentration on the multichannel structure of MCCGs was investigated by using confocal laser scanning microscopy along with fluorescent staining. The channel diameter increased with increasing NaCl concentrations in the collagen solution and the phosphate buffer solution. In contrast, the channel number decreased with increasing NaCl concentrations. Engineered tissues with various lumen-like structures were constructed by seeding and culturing Madin-Darby canine kidney cells on MCCGs. The diameter of the lumen and the number of lumens per unit area were controllable by regulating the multichannel structure of cylindrical MCCG. We believe that our methodology for the construction of engineered tissues possessing epithelial lumen-like structures will prove helpful in regeneration of giant tissues with various hierarchical structures.

DOI： 10.1021/acsbiomaterials.5b00003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Wnt signaling pathways are tightly regulated by ubiquitination, and dysregulation of these pathways promotes tumorigenesis. It has been reported that the ubiquitin ligase RNF43 plays an important role in frizzled-dependent regulation of the Wnt/beta-catenin pathway. Here, we show that RNF43 suppresses both Wnt/beta-catenin signaling and noncanonical Wnt signaling by distinct mechanisms. The suppression of Wnt/beta-catenin signaling requires interaction between the extracellular protease-associated (PA) domain and the cysteine-rich domain (CRD) of frizzled and the intracellular RING finger domain of RNF43. In contrast, these N-terminal domains of RNF43 are not required for inhibition of noncanonical Wnt signaling, but interaction between the C-terminal cytoplasmic region of RNF43 and the PDZ domain of dishevelled is essential for this suppression. We further show the mechanism by which missense mutations in the extracellular portion of RNF43 identified in patients with tumors activate Wnt/beta-catenin signaling. Missense mutations of RNF43 change their localization from the endosome to the endoplasmic reticulum (ER), resulting in the failure of frizzled-dependent suppression of Wnt/beta-catenin signaling. However, these mutants retain the ability to suppress noncanonical Wnt signaling, probably due to interaction with dishevelled. RNF43 is also one of the potential target genes of Wnt/beta-catenin signaling. Our results reveal the molecular role of RNF43 and provide an insight into tumorigenesis.

DOI： 10.1128/MCB.00159-15

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The effects of a flow field on the amyloid fibrillogenesis of beta-lactoglobulin (beta LG) were investigated using a flow birefringence method and AFM imaging experiments. A 4 wt% beta LG aqueous solution was incubated at pH 2 and 80 degrees C. A flow field was then applied by stirring at 250 and 474 rpm. An incubation without stirring was used as a control sample. Flow birefringence measurements were taken at room temperature from the incubated sample solutions in which an elongational flow field was used. The birefringence pattern obtained indicated that the fibrils formed by the incubation were rigid rod-like molecules. Birefringence relaxation experiments revealed at least two relaxation processes, suggesting a double peaked distribution function for fibrils length. The length distribution of fibrils expected from the birefringence experiments was confirmed by the AFM images of amyloid fibrils. The order of the expected length of the resultant fibrils in both longer and shorter length distributions was those stirred at 250 rpm congruent to 474 rpm > 0 rpm. The effects of the flow field applied during the incubation on amyloid fibrillogenesis was discussed on the basis of the rate process consideration. The present results demonstrated that the flow field should be considered as an important factor that regulates the fibrillogenesis of globular proteins. (C) 2014 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ijbiomac.2014.06.034

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：IEEE  

To control the structure of collagen gel with multichannel structure (Multi-Channel Collagen Gel: MCCG), the effect of gelation temperature on the multichannel structure of MCCG have been investigated. The diameter of channel decreased with increasing gelation temperature. By contrast, the number of channel increased with increasing the gelation temperature. The results showed that the multi-channel structure of MCCG can be controlled by regulating the gelation temperature.

DOI： 10.1109/MHS.2014.7006141

DOI： 10.1109/mhs.2014.7006141

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Mimicking the complicated anisotropic structures of a native tissue is extremely important in tissue engineering. In a previous study, we developed an anisotropic collagen gel scaffold (ACGS) having a hierarchical structure and a properties gradient. In this study, our objective was to see how cells remodel the scaffolds through the cells-ACGS interaction. For this purpose, we cultured osteoblastic cells on ACGS, which we regarded as a model system for the cells-extracellular matrix (cell-ECM) interaction. Changes in the ACGS-cell composites structure by cell-ECM interactions was investigated from a macroscopic level to a microscopic level. Osteoblastic cells were also cultured on an isotropic collagen gel (ICGS) as a control. During the cultivation, mechanical stimuli were applied to collagen-cell composites for adequate matrix remodeling. Confocal laser scanning microscope (CLSM) was used to observe macroscopic changes in the ACGS-cell composite structure by osteoblastic cells. Small-angle X-ray scattering (SAXS) measurements were performed to characterize microscopic structural changes in the composites. Macroscopic observations using CLSM revealed that osteoblastic cells remained only in the diluted phase in ACGS and they collected collagen fibrils or formed a toroidal structure, depending on the depth from the ACGS surface in the tubular diluted phase. The cells were uniformly distributed in ICGS. SAXS analysis suggests that collagen fibrils were remodeled by osteoblastic cells, and this remodeling process would be affected by the structure difference between ACGS and ICGS. These results suggest that we directly regulate cell-ECM interaction by the unique anisotropic and hierarchical structure of ACGS. The cell-gel composite presented in this study would promise an efficient scaffold material in tissue engineering

DOI： 10.1021/am303254e

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

We have studied stress relaxation of bovine femoral cortical bone specimens treated with KOH aqueous solution which had been known to degrade selectively protein molecules in bone. With the KOH treatment, we found an increase in specimens' volume. This increase was regarded as swelling of the bone specimen, presumably due to matrix protein network degradation including that of collagen. In an analogy of bone to gel structure, an increasing ratio of specimen volume was used as an indicating parameter for the matrix protein network degradation by the treatment. Although an empirical equation with a linearly combined form of two Kohlrausch-Williams-Watts (KWW) functions has been shown to describe the stress relaxation of bone specimens, a single KWW function was suitable for the bone specimens treated with KOH solution for as little as 3 h. In KOH treated specimens, both the initial modulus and the relaxation time decreased with the volume-increasing ratio, while the relaxation time distribution did not change. A chemo-rheological consideration attributed the reduction of modulus values to the network degradation in the organic matrix phase. The relaxation time of KOH treated specimens was thought to be related to the longer relaxation time of untreated bones, although there was a discontinuity between the extrapolated relaxation time values for KOH treated specimens and untreated specimens. This discontinuity may have originated from the release of residual stress existing in the bone by the matrix protein degradation. The results of the present study suggest that the state of matrix protein is crucial for integrating the mechanical properties of bone. (c) 2012 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jbiomech.2012.11.038

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：日本バイオレオロジー学会  

The viscoelastic properties of cell-seeded agarose gel were measured as a function of culture time. Because the seeded cells, MC3T3-E1, were osteoblast-like cells, the system can be regarded as a model osteogenesis system. For all specimens the characteristic stress relaxation curve of agarose gel was observed-a large relaxation up to 104 s followed by a gel plateau, where the former was attributed to molecular motion of polymer chains between two adjacent cross-links of the gel and the latter to the elasticity of the gel network. The viscoelasticity was quantified by fitting stress relaxation data to an empirical equation. The relaxation time and its distribution did not change with culture time. The initial and equilibrium moduli, E0 and Ee, respectively, and relaxation strength, ΔE = E0 - Ee, did not change up to day 15 of culture but changed significantly at day 18 of culture. The change in ΔE with culture period correlated well with that in E0. The changes in the mechanical properties of the cell-seeded agarose gel system were explained in terms of the function of MC3T3-E1 in precipitating calcium phosphate mineral particles. The precipitation was detected by alizarin red S staining of the system at day 9 of culture. The precipitated calcium phosphate was confirmed to be hydroxyapatite (HAp) and the amount of HAp increased monotonically with culturing time, both of which were observed by X-ray diffraction studies. The dependence of the modulus of the composite on mineral fraction is discussed. A simple model of mixing of the components based on the continuum material concept was not applicable, but a model considering percolation of mineral particles in a network chain with culture time was suitable to explain the observed results. The results may be particularly important for predicting the stiffness of functionally engineered bony tissue implanted in a fractured bone. © 2011 Japanese Society of Biorheology.

DOI： 10.1007/s12573-011-0043-2

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Authorship：Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：U B C PRESS  

Chemokines play a crucial role in developmental processes and recent studies have revealed that they also control gastrulation movements. In this paper, we report the expression patterns of xSDF-l alpha, xCXCR4 and xCXCR7 and regulation of the expression of xSDF-la and xCXCR4 during gastrulation. We performed whole mount in situ hybridization (WISH) and quantitative realtime RT-PCR (qRT-PCR) analyses to examine the distribution of transcripts. The effect of activin/nodal signaling on the expression of xSDP-1 alpha and its receptors was examined by animal cap assay and microinjection of cer-s mRNA. We have demonstrated that the xSDF-la transcript is increased in the blastocoel roof during gastrulation, but not in the involuted mesoderm. xCXCR4 was expressed in the mesendoderm at late blastula and was retained throughout gastrulation. xCXCR7 was found in the dorsal lip around the blastopore in the early gastrula stage and became localized in the presumptive notochord later. We also show that the expression of xCXCR4 and xSDF-l alpha were reciprocally regulated by activin/nodal signaling. These results suggest that xSDP-1 alpha and its receptors contribute to the cell arrangement of mesoderm cells and their expression patterns are partially regulated by activin/nodal signaling.

DOI： 10.1387/ijdb.120130af

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：14th International Xenopus conference Organizing Committee  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

We have found that dialysis of S mg/mL collagen solution into the phosphate solution with a pH of 7.1 and an ionic strength of 256 mM at 25 degrees C results in a collagen gel with a birefringence and tubular pores aligned parallel to the growth direction of the gel. The time course of averaged diameter of tubular pores during the anisotropic gelation was expressed by a power law with an exponent of 1/3, suggesting that the formation of tubular pores is attributed to a spinodal decomposition-like phase separation. Small angle light scattering patterns and high resolution confocal laser scanning microscope images of the anisotropic collagen gel suggested that collagen fibrils are aligned perpendicular to the growth direction of the gel. The positional dependence of the order parameter of the collagen fibrils showed that the anisotropic collagen gel has an orientation gradient.

DOI： 10.1021/bm200869p

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Language：Japanese   Publisher：The Japan Society of Mechanical Engineers  

DOI： 10.1299/jsmebio.2012.24._7b32-1_

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：The Materials Research Society of Japan  

DOI： 10.14723/tmrsj.36.387

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

A transcriptional corepressor, Xenopus furry (Xfurry), is expressed in the chordamesodermal region and induces secondary dorsal axes when overexpressed on the ventral side of the embryo. The N-terminal furry domain functions as a repressor, and the C-terminal leucine zipper (LZ) motifs /coiled-coil structure, found only in vertebrate homologs, contributes to the nuclear localization. The engrailed repressor (enR)+LZ repressor construct, which has properties similar to Xfurry, induced several chordamesodermal genes. In contrast, an antisense morpholino oligonucleotide, Xfurry-MO, and the activating construct, herpes simplex virus protein (VP16)+LZ, had effects opposite those of Xfurry overexpression. Because blocking protein synthesis with cycloheximide superinduced several Xfurry transcriptional targets, and because expression of enR+LZ induced such genes under cycloheximide treatment, we analyzed the role of an Xfurry transcriptional target, microRNA miR-15. Cycloheximide reduced the expression of primary miR-15 (pri-miR-15), whereas miR-15 reduced the expression of genes superinduced by cycloheximide treatment. These results show that Xfurry regulates chordamesodermal genes by contributing to repression of pretranscriptional gene silencing by miR-15.

DOI： 10.1073/pnas.1008954107

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Brachyury, a member of the T-box transcription family identified in a diverse array of metazoans, was initially recognized for its function in mesoderm formation and notochord differentiation in vertebrates: however, its ancestral role has been suggested to be in control of morphogenetic movements. Here, we show that morpholino oligonucleotide knockdown of Brachyury (MlBra) in embryos of a ctenophore, one of the most ancient groups of animals, prevents the invagination of MlBra expressing stomodeal cells and is rescued with corresponding RNA injections. Injection of RNA encoding a dominant-interfering construct of MlBra causes identical phenotypes to that of RNA encoding a dominant-interfering form of Xenopus Brachyury (Xbra) in Xenopus embryos. Both injected embryos down-regulate Xbra downstream genes, Xbra itself and Xwnt11 but not axial mesodermal markers, resulting in failure to complete gastrulation due to loss of convergent extension movements. Moreover, animal cap assay reveals that MlBra induces Xwnt11 like Xbra. Overall results using Xenopus embryos show that these two genes are functionally interchangeable. These functional experiments demonstrate for the first time in a basal metazoan that the primitive role of Brachyury is to regulate morphogenetic movements, rather than to specify endomesodermal fates, and the role is conserved between non-bilaterian metazoans and vertebrates. (C) 2009 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2009.12.019

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Although physiological saline (0.15 M NaCl aqueous solution) has been used as a storage Solution to prevent bone specimens from drying, there have been reports that Ca(2+) ions dissolve front bone specimens during the storage in saline. In order to determine whether Such storage has a marked effect oil mechanical properties, the relaxation Modulus of bovine cortical bone stored in physiological saline was compared with that stored in a buffer solution containing a Sufficient amount of Ca(2+) ions. After storage in saline, the modulus Value of specimens was significantly reduced from that before storage. Oil the other hand, the modulus value of specimens soaked in the solution containing Sufficient Ca(2+) ions did not change after storage. The relaxation rate of a bone specimen stored in physiological saline was larger than that of a specimen stored in Ca(2+)-buffered saline solution and that of the control specimens. The results Suggest that by the dissolution of Ca(2+) ions from a bone specimen during storage in physiological saline, percolated paths of mineral phase and of reinforced matrix phase are disjoined, resulting ill reduction in the elastic modulus and change in the viscoelastic properties of bone. (C) 2008 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jbiomech.2008.09.019

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Hyaluronan matrix plays an important role during vertebrate cardiogenesis. Transcripts for the hyaluronan synthase Has2 gene are expressed in heart anlage, and disruption of either Has2 or versican, a hyaluronan matrix component, abrogates normal cardiac morphogenesis. However, the mechanisms by which hyaluronan matrix contributes to early heart development are largely unknown. Here we show that Xenopus hyaluronan and proteoglycan-binding link protein 3 (XHAPLN3) helps to maintain hyaluronan matrix around the cardiac anlage, and thereby contribute to cardiogenesis. XHAPLN3 mRNA transcript localization overlapped with the mRNA expression of both Xhas2 and Xversican at the heart anlage of early tailbud (stage 23) embryos. Furthermore, knockdown of XHAPLN3 or Xhas2 with morpholino antisense oligos caused a heart deficiency in developing tadpoles. Our results show when and how components of the hyaluronan matrix function in cardiogenesis, improving our understanding of how extracellular matrix participates in embryogenesis. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2008.03.042

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本生物物理学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PHARMACEUTICAL SOC JAPAN  

Treatment of Xenopus blastula with activin converts undifferentiated presumptive ectoderm (animal cap) into mesoderm and endoderm in a dose-dependent manner. At low concentrations, activin induces ventral mesoderm such as blood cells. Here we show that activin-treated aggregates of animal cap cells prepared from undifferentiated presumptive ectoderm and transplanted into Xenopus embryos differentiated to form red blood cells and vascular endothelial cells. We compared gene expression profiles of the activin-treated with untreated aggregates of animal cap cells using microarray analysis. This revealed 838 clones including vascular-related genes that were expressed at levels at least 2-fold greater in the activin-treated aggregates than in the untreated controls. Of these, 356 were known Xenopus genes, 296 had homologues, and 186 were unknown genes. These findings identified novel vascular-related genes and provided insights into how the blood vessel system establishes in normal development.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

The aim of this study was to determine the difference between hydrodynamic properties of DNA-cetyltrimethylammonium (CTA) complex and those of DNA, which may be related to the difference in fibre-forming ability of DNA-CTA from that of DNA. Responses of DNA and DNA-CTA complex to an elongational flow field were investigated. In both solution systems, results suggesting a coil-stretch transition were obtained. From a critical strain rate value, the radius of gyration of DNA-CTA molecules in ethanol-glycerol solution was revealed to be 0.3-0.5 times of that of DNA in aqueous NaCl solution. Shear viscosity of DNA-CTA solution was much smaller than that of DNA solution, also suggesting a smaller size of DNA-CTA in ethanol-glycerol solution than that of DNA in aqueous NaCl solution. The plateau birefringence value of the DNA-CTA system, a parameter that indicates the local molecular conformation and the molecular arrangement, was only about 1/10 of that of the DNA system. There is an empirically determined molecular model of DNA-CTA complex in which a DNA molecule is sheathed by a cylindrical crust made of CTA chains. This structure reduces the DNA molecular density in a pure elongational flow field region but cannot explain the observed reduction of birefringence intensity. The small plateau birefringence value of DNA-CTA compared with that of DNA was attributed to the reduced molecular polarizability by the particular conformation of DNA molecules and CTA chains in the DNA-CTA system such as that expected by the conformational models. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.ijbiomac.2006.08.016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Activin-like signaling plays an important role in early embryogenesis. Activin A, a TGF beta family protein, induces mesodermal/endodermal tissues in animal cap assays. In a screen for genes expressed early after treatment with activin A, we isolated a novel gene, denoted as BENI (Brachyury Expression Nuclear Inhibitor). The BENI protein has a conserved domain at the N-terminus that contains a nuclear localization signal (NLS), and two other NLSs in the C-terminal domain. BENImRNA was localized to the animal hemisphere at the gastrula stages and to ectoderm except for neural regions at stage 17; expression persisted until the tadpole stage. The overexpression of BENI caused gastrulation defects and inhibition of elongation of activin-treated animal caps with reduction of Xbra expression. Moreover, whole-mount in situ hybridization revealed reduced expression of Xbra in BENI mRNA-injected regions of gastrula embryos. Functional knockdown of BENI using an antisense morpholino oligonucleotide also resulted in an abnormal phenotype of embryos curling to the dorsal side, and excessive elongation of activin-treated animal caps without altered expression of mesodermal markers. These results suggested that BENI expression is regulated by activin-like signaling, and that this regulation is crucial for Xbra expression. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2006.11.014

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

The cardiovascular development is the elaborate process, and despite the extensive studies, the mechanisms underlying endothelial, hematopoietic, and cardiac developments, as well as the interrelation between these processes, are not fully understood. In this study, we demonstrated that Xenopus apelin and Xmsr play pivotal roles in cardiovascular development. Apelin is a recently identified ligand for an orphan G-protein-coupled receptor APJ and is involved in fluid homeostasis in mammals. Xenopus preproapelin (Xpreproapelin) was isolated and its mRNA localized to the region around the presumptive blood vessels, which are overlapping or adjacent to those expressing Xmsr, the Xenopus homologue of APJ Overexpression of Xpreproapelin disorganized the expression of the endothelial precursor cell marker XlFli and the hematopoietic precursor cell marker SCL at the neurula, whereas embryos injected with morpholino antisense oligonucleotides for Xapelin and Xmsr displayed attenuated expression of Tie2, alpha-globin, XPOX2, and cTnI, markers of endothelium, erythrocytes, myeloid cells, and cardiomyocytes, respectively. XlFli morpholino had similar effects to Xapelin and Xmsr morpholinos on cardiac differentiation, suggesting an unexpected potential relationship between the endothelium and cardiac differentiation. Forced expression of constitutive active G alpha i rescued the phenotypes of Xmsr morpholino-injected embryos, indicating that the i/o type of G protein alpha subunit acts downstream of Xmsr. (c) 2006 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2006.06.028

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Wnt signaling pathways are essential in various developmental processes including differentiation, proliferation, cell migration, and cell polarity. Wnt proteins execute their multiple functions by activating distinct intracellular signaling cascades, although the mechanisms underlying this activation are not fully understood. We identified a novel Daple-like protein in Xenopus and named it xDal (Xenopus Daple-like). As with Daple, xDal contains several leucine zipper-like regions (LZLs) and a putative PDZ domain-binding motif, and can interact directly with the dishevelled protein. In contrast to mDaple, injection of xDal mRNA into the dorso-vegetal blastomere does not induce ventralization and acted synergistically with xdsh in secondary axis induction. XDal also induced expression of siamois and xnr-3, suggesting that XDal functions as a positive regulator of the Wnt/-catenin pathway. Injection of xDal mRNA into the dorso-animal blastomere, however, induced gastrulation-defective phenotypes in a dose-dependent manner. In addition, xDal inhibited activin-induced elongation of animal caps and enhanced c-jun phosphorylation. Based on these findings, xDal is also thought to function in the Wnt/JNK pathway. Moreover, functional domain analysis with several deletion mutants indicated that xDaI requires both a putative PDZ domain-binding motif and at least one LZL for its activity. These findings with xDal will provide new information on the Wnt signaling pathways. 2005 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.mod.2005.05.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Activin is a potent inducer of mesoderm in amphibian embryos. We previously reported that low concentrations of activin could induce the formation of blood cells from Xenopus explants (animal caps). Both hematopoietic and vascular endothelial cell lineages are believed to share a common precursor, termed hemangioblasts. In this study, we tried to induce differentiation of vascular endothelial cells in aggregates derived from Xenopus animal caps. Aggregates formed from cells that were co-treated with activin and angiopoietin-2 expressed the vascular endothelial markers, X-msr, Xtie2 and Xegf17. However, none of these aggregates expressed the hematopoietic marker genes, globin alpha T3, alpha T5, alpha A or GATA-1. We used microarray analysis to compare the gene expression profiles of aggregates treated with activin alone or with activin and angiopoietin. The combination, but not activin alone, induced expression of vascular-related genes such as XI-fli and VEGF. These results demonstrate that treatment of dissociated animal cap cells with activin and angiopoietin-2 can induce differentiation of endothelial cells, and provides a promising model system for the in vitro study of blood vessel induction in vertebrates.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PERGAMON-ELSEVIER SCIENCE LTD  

Here, we report changes gene expression and morphology of the renal epithelial cell line, A6, which was derived from Xenopus laevis adult kidney that had been induced by long-term culturing with a three-dimensional clinostat. An oligo microarray analysis on the A6 cells showed that mRNA levels for 52 out of 8091 genes were significantly altered in response to clinorotation. On day 5, there was no dramatic change in expression level, but by day 8 and day 10, either upregulation or downregulation of gene expression became evident. By day 15, the expression levels of 18 out of 52 genes had returned to the original levels, while the remaining 34 genes maintained the altered levels of expression. Quantitative analyses of gene expression by real-time PCR confirmed that changes in the mRNA levels of selected genes were found only under clinorotation and not under hypergravity (7g) or ground control. Morphological changes including loss of dome-like structures and disorganization of both E-cadherin adherence junctions and cortical actin were also observed after 10 days of culturing with clinorotation. These results revealed that the expression of selected genes was altered specifically in A6 cells cultured under clinorotation. (c) 2005 COSPAR. Published by Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.asr.2005.04.100

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Language：English   Publishing type：Research paper (scientific journal)  

We documented changes in morphology and gene expression of the renal epithelial cell line A6 derived from Xenopus leavis adult kidney induced by long-term culturing with three dimensional clinostats. An oligo microarray analysis on A6 cells showed that mRNA levels of 52 out of 8091 genes were significantly altered in response to clinorotation. Upregulation or downregulation of gene expression became evident on day 8 and day 10 while there was no significant change on day 5. However, on day 15, expression of 18 out of 52 genes resumed to the levels similar to its original levels while remaining 33 genes maintained altered levels of expression. Quantitative analyses of gene expression by real-time PCR confirmed that changes in mRNA levels of selected genes were found only under clinorotation but not under hypergravity (7 G) and ground control (1 G). Morphological changes including loss of dome-like structures, disassembly of E-cadherin adherence junctions and disassembly of cortical actin were also observed over 10 days of culturing with clinorotation. The results revealed genes which expression was altered specifically in A6 cells cultured under clinorotation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：10th International Xenopus Meeting Organizing Committee  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING LTD  

Small Ubiqutin-related modifier (SUMO), which is responsible for the ubiquitination-like post-translational modification 'sumoylation', regulates a number of biological processes including, in particular, transcription. The rat protein Axam, which possesses SUMO-specific protease activity, was shown to inhibit the Wnt signalling pathway. Several other components of the pathway are also sumoylated, so the mechanism of this modification has itself been linked to Wnt signalling. However, the functional interactions between SUMO and Wnt signalling are not well understood. This study identified a novel SUMO-specific protease in Xenopus, which was denoted XSENP1. The C-terminus of XSENP1 is highly conserved across the SUMO-specific protease family, and in vitro XSENP1 possesses hydrolase and desumoylation activity. Over-expression of XSENP1 in vivo inhibited dorso-anterior development of Xenopus embryos and suppressed Wnt signalling target gene expression in a manner similar to Axam. Deletion analysis of XSENP1 showed that inhibition of the Wnt signalling pathway requires protease activity. Moreover, XSENP1 inhibits ectopic axis induction by Dvl, beta-catenin and the constitutively active form of beta-catenin, but not by siamois. These results indicate that the dorsal expression of XSENP1 obstructs head development in Xenopus laevis and that this effect may result from inhibition of the canonical Wnt pathway downstream of beta-catenin, but upstream of siamois.

DOI： 10.1111/j.1365-2443.2004.00757.x

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-LISS  

Wnt signaling pathways are involved during various stages in the development of many species. In Xenopus, the accumulation of beta-catenin on the dorsal side of embryo is required for induction of the organizer, while the head structure formation requires inhibition of Wnt signaling. Here, we report a role for xldax, a negative regulator of Wnt signaling. Xldax is expressed in neural tissues at the neurula stage, and in the restricted region of the tadpole brain. Ectopic expression of xldax inhibits the target gene expression, suggesting that xldax can inhibit canonical Wnt signaling. To examine the function of xldax, a morpholino oligo for xldax (xldaxMO) was designed. An injection into an animal pole cell caused a loss of forebrain. The anterior neural marker expression is decreased in xldaxMO-injected embryo, suggesting that xldax is required for anterior neural development. Moreover, a negative regulator that acts downstream of xldax rescued this defect. We propose that Idax functions are dependent on the canonical Wnt pathway and are crucial for the anterior neural development. (C) 2004 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.20037

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Language：English   Publisher：The 6th conference Asia-Pacific International Molecular Biology Network Organizing Committee  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

NDRG1 is a member of the N-myc downstream-regulated gene (NDRG) family and is involved in cellular differentiation, activation of p53, cell cycle arrest, metastasis, and hypoxia. Expression of NDRG1 is repressed by the proto-oncogene, N-myc during mouse development, although the exact functional role of NDRG1 in development remains unknown. Here, we report the characterization of Xenopus laevis NDRG1 (xNDRG1) during X laevis development. Expression of xNDRG1 transcript was first detected at stage 15, and was localized to the presumptive pronephric anlagen at stage 26 and to pronephros, eye, branchial arches, and tail-bud at stage 32. Overexpression of xNDRG1 results in a reduced pronephros and disorganized somites. Depletion of xNDRG1, using morpholinos, causes failure of pronephros development. These results suggest that xNDRG1 is required for pronephros development in X. laevis. (C) 2003 Elsevier Inc. All rights reserved.

DOI： 10.1016/S0006-291X(03)01522-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING ASIA  

Studies have shown that the activin type IB receptor is specific for activin/nodal signaling. Activin is produced by follicle cells in the ovary, and is incorporated into the oocytes. Antisera against three peptides were prepared, encompassing the extracellular, intracellular and serine/threonine kinase domains of the Xenopus type IB activin receptor (XALK4). Immunocytochemistry was done using these antisera to investigate the distribution of XALK4 in the Xenopus ovary. All three antisera stained the mitochondrial cloud of Xenopus previtellogenic oocytes. Purified antibody against the intracellular domain also recognized the mitochondrial cloud. Immunoelectron microscopy localized XALK4 on the endoplasmic reticulum of the mitochondrial cloud, although not on mitochondria.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Axam has been identified as I novel Axin-binding protein that inhibits the Wnt signaling pathway. We studied the molecular mechanism by which Axam stimulates the downregulation of beta-catenin. The C-terminal region of Axam has an amino acid sequence similar to that of the catalytic region of SENP1, a SUMO-specific protease (desumoylation enzyme), Indeed, Axam exhibited activity to remove SUMO from sumoylated proteins in vitro and in intact cells. The Axin-binding domain is located in the central region of Axam, which is different from the catalytic domain. Neither the Axin-binding domain nor the catalytic domain alone was sufficient for the downregulation of beta-catenin. An Axam fragment which contains both domains was able to decrease the level of beta-catenin. On substitution of Ser for Cys(547) in the catalytic domain, Axam lost its desumoylation activity. Further, this Axam mutant decreased the activity to downregulate beta-catenin. Although Axam strongly inhibited axis formation and expression of siamois, a Wnt-response gene, in Xenopus embryos, Axam(C547S) showed weak activities. These results demonstrate that Axam functions as a desumoylation enzyme to downregulate beta-catenin and suggest that sumoylation is involved in the regulation of the Wnt signaling pathway.

DOI： 10.1128/MCB.22.11.3803-3819.2002

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING ASIA  

Recent studies suggest that extra-embryonic tissues may be essential sources of early organizing signals for the mouse embryo. In vitro studies of human amniotic epithelial cells (HAEC) have shown that the amnion can produce various biologically active substances. In this study, the synthesis and release of activin A and noggin, and the activin signaling pathway, was investigated in HAEC. Conditioned medium from cultured HAEC contained activin A which was functionally active in Xenopus laevis animal cap assays. Immunohistochemistry, western blotting and reverse transcription-polymerase chain reaction confirmed that HAEC also synthesize and release noggin. Noggin transcripts were induced by the addition of recombinant activin A, and activin A was inhibited by activin antibody except in the presence of cycloheximide (CHX). These data demonstrate that noggin mRNA expression is induced directly by activin A without new protein synthesis, indicating that noggin is a primary response gene. The results suggest that there is an activin signaling pathway in HAEC, and that the human amnion might therefore be involved in neural formation during early development.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Duplin binds to beta-catenin and inhibits the Wnt signaling pathway, thereby leading to repression of the beta-catenin-mediated transactivation and Xenopus axis formation. To find an additional function of Duplin, yeast two-hybrid screening was carried out. Importin a was isolated as a binding protein of Duplin. Importin a bound directly to basic amino acid clusters of Duplin. Although Duplin was present in the nucleus, deletion of the basic amino acid clusters (Duplin(Delta500-584)) retained Duplin in the cytoplasm. Duplin(Delta500-584) bound to beta-catenin as efficiently as wild-type Duplin, but it neither repressed Wnt-dependent Tcf transcriptional activation in mammalian cells nor showed ventralization in Xenopus embryos. The Duplin mutant without a beta-catenin-binding region lost the ability to inhibit the Wnt-dependent Tcf activation, but retained its ventralizing activity. Furthermore, Duplin not only suppressed beta-catenin-dependent axis duplication and expression of siamois, a Wnt-regulated gene, but also inhibited siamois-dependent axis duplication. These results indicate that Duplin is translocated to the nucleus by interacting with importin a, and that nuclear localization is essential for the function of Duplin. Moreover, Duplin has an additional activity of inhibiting the Wnt signaling pathway by affecting the downstream beta-catenin target genes.

DOI： 10.1074/jbc.M108433200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

BMP-4 has been implicated in the patterning of the Dorsal-Ventral axis of mesoderm and ectoderm. In this study, we describe the posteriorizing effect of BMP-4 on the neural inducing ability of dorsal mesoderm (dorsal lip region) in Xenopus gastrulae. Dorsal lip explants dissected from stage 10.25 embryos retained anterior inducing ability when precultured for 6 hrs until sibling embryos reach stage 12. When the dorsal lips from stage 10.25 embryos were treated with a range of BMP-4 concentrations, posterior tissues were induced in adjacent ectoderm in a dose-dependent manner. Thus activin-treated explants able to act as head inducers can also induce posterior structures in the presence of BMP-4. To investigate whether BMP-4 directly affects the inducing ability of dorsal mesoderm, we blocked the BMP4 signaling pathway by injection of mRNA encoding a truncated form of the BMP-4 receptor (tBR) mRNA. Under these conditions, activin-treated explants induced anterior tissues following BMP-4 treatment. Taken together, these results indicate that BMP-4 may affect the head inducing ability of dorsal mesoderm and confer trunk-tail inducing ability during Xenopus gastrulation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Although casein kinase I epsilon (CKI epsilon) has been shown to regulate the Wnt signaling pathway positively, its mode of action is not clear. In this study we show that CKI epsilon activates the Wnt signaling pathway in co-operation with Dv1. CKI epsilon and Axin associated with different sites of Dv1, and CKI epsilon and Dv1 interacted with distinct regions on Axin. Therefore, these three proteins formed a ternary complex. Either low expression of Dv1 or CKI epsilon alone did not accumulate beta -catenin, but their co-expression accumulated greatly. Dv1 and CKI epsilon activated the transcriptional activity of T cell factor (Tcf) synergistically. Although the Dv1 mutant that binds to Axin but not to CKI epsilon activated Tcf, it did not synergize with CKI epsilon Another Dv1 mutant that does not bind to Axin did not activate Tcf irrespective of the presence of CKI epsilon Furthermore, Dv1 and CKI epsilon co-operatively induced axis duplication of Xenopus embryos. These results indicate that Dv1 and CKI epsilon synergistically activated the Writ signaling pathway and that the binding of the complex of Dv1 and CKI epsilon to Axin is necessary for their synergistic action.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Axin, a negative regulator of the Wnt signaling pathway, forms a complex with glycogen synthase kinase-3 beta (GSK-3 beta), beta -catenin, adenomatous polyposis coli (APC) gene product, and Dvl, and it regulates GSK-3 beta -dependent phosphorylation in the complex and the stability of beta -catenin. Using yeast two-hybrid screening, we found that regulatory subunits of protein phosphatase 2A, PR61 beta and -gamma, interact with Axin. PR61 beta or -gamma formed a complex with Axin in intact cells, and their interaction was direct. The binding site of PR61 beta on Axin was different from those of GSK-3 beta, beta -catenin, APC, and Dvl. Although PR61 beta did not affect the stability of beta -catenin, it inhibited Dvl- and beta -catenin-dependent T cell factor activation in mammalian cells. Moreover, it suppressed beta -catenin-induced axis formation and expression of siamois, a Wnt target gene, in Xenopus embryos, suggesting that PR61 beta acts either at the level of beta -catenin or downstream of it. Taken together with the previous observations that PR61 interacts with APC and functions upstream of beta -catenin, these results demonstrate that PR61 regulates the Wnt signaling pathway at various steps.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

In attempting to clarify the roles of Dvl in the Wnt signaling pathway, we identified a novel protein which binds to the PDZ domain of Dvl and named it Idax (for inhibition of the Dvl and Axin complex). Idax and Axin competed with each other for the binding to Dvl. Immunocytochemical analyses showed that Idax was localized to the same place as DvI in cells and that expression of Axin inhibited the colocalization of Dvl and Idax. Further, Wnt-induced accumulation of beta -catenin and activation of T-cell factor in mammalian cells were suppressed by expression of Idax. Expression of Idax in Xenopus embryos induced ventralization with a reduction in the expression of siamois, a Wnt-inducible gene. Idax inhibited Wnt- and Dvl- but not beta -catenin-induced axis duplication. It is known that Dvl is a positive regulator in the Wnt signaling pathway and that the PDZ domain is important for this activity. Therefore, these results suggest that Idax functions as a negative regulator of the Wnt signaling pathway by directly binding to the PDZ domain of Dvl.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：COMPANY OF BIOLOGISTS LTD  

The asymmetric distribution of cellular components is an important clue for understanding cell fate decision during embryonic patterning and cell functioning after differentiation. In C, elegans embryos, PAR-3 and aPKC form a complex that colocalizes to the anterior periphery of the one-cell embryo, and are indispensable for anterior-posterior polarity that is formed prior to asymmetric cell division. In mammals, ASIP (PAR-3 homologue) and aPKC lambda form a complex and colocalize to the epithelial tight junctions, which play critical roles in epithelial cell polarity. Although the mechanism by which PAR-3/ASIP and aPKC regulate cell polarization remains to be clarified, evolutionary conservation of the PAR-3/ASIP-aPKC complex suggests their general role in cell polarity organization. Here, we show the presence of the protein complex in Xenopus laevis, In epithelial cells, XASIP and XaPKC colocalize to the cell-cell contact region. To our surprise, they also colocalize to the animal hemisphere of mature oocytes, whereas they localize uniformly in immature oocytes. Moreover, hormonal stimulation of immature oocytes results in a change in the distribution of XaPKC 2-3 hours after the completion of germinal vesicle breakdown, which requires the kinase activity of aPKC, These results suggest that meiotic maturation induces the animal-vegetal asymmetry of aPKC.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3 beta (GSK-3 beta), beta -catenin, DvI, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3 beta efficiently phosphorylates beta -catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axin associating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of DvI with Axin and the activity of DvI to suppress GSK-3 beta -dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of beta -catenin in SW480 cells and inhibited Wnt-dependent axis duplication in Xenopus embryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of DvI to Axin.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

beta -Catenin is efficiently phosphorylated by glycogen synthase kinase-3 beta in the Axin complex in the cytoplasm, resulting in the down-regulation. In response to Wnt, beta -catenin is stabilized and translocated into the nucleus where it stimulates gene expression through Tcf/Lef, Here we report a novel protein, designated Duplin (for axis duplication inhibitor), which negatively regulates the function of beta -catenin in the nucleus. Duplin was located in the nucleus. Duplin bound directly to the Armadillo repeats of beta -catenin, thereby inhibiting the binding of Tcf to beta -catenin. It did not affect the stability of beta -catenin but inhibited Wnt- or beta -catenin-dependent Tcf activation. Furthermore, expression of Duplin in Xenopus embryos inhibited the axis formation and beta -catenin-dependent axis duplication, and prevented the beta -catenin's ability to rescue ventralizing phenotypes induced by ultraviolet light irradiation. Thus, Duplin is a nuclear protein that inhibits beta -catenin signaling.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE ASIA  

Wnt signaling plays an important role in axis formation in early vertebrate development. Axin is one Wnt signaling regulator that inhibits this pathway. The effects of the injection of mRNA of several rat Axin (rAxin) mutants on axis formation in Xenopus embryos were examined. It was found that rAxin mutants containing only a regulation of G-protein signaling (RGS) domain fragment or with deletion of the RGS domain induced axis formation. Because the RGS domain is a major adenomatous polyposis coli gene product (APC)-binding domain, APC association with glycogen synthase kinase 3 beta (GSK3 beta) on the Axin molecule may be important in inhibition of axis formation. The ventralizing activities of wild-type rAxin and a mutant in which the Dishevelled and Axin (DIX) domain was deleted (Delta DIX mutant) were examined. Histological examination and gene expression revealed that the ventralizing activity of the Delta DIX mutant was weaker than that of wild-type rAxin. This finding suggests that the C-terminus of rAxin contributes to the inhibition of Wnt signaling in Xenopus embryos. Furthermore, an rAxin mutant that contained both the RGS and GSK3 beta -binding domains affected both the dorsal and ventral sides of blastomeres, mediated ectodermal fate and induced expansion of notochord and/or endoderm, but did not induce axis formation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD  

Teratomas are rare in amphibians and the neoplasm described here, which had a significant thyroid carcinoma component, is the first tumour of this type to be reported in Xenopus laevis. The thyroid component contained moderately to well-differentiated acinar glands showing much hyperplasia, dysplasia, and reduced and distorted colloid reservoirs. Cartilaginous, neural, muscular, mesenchymal and gut-like epithelial components were also observed in this ventral mediastinal neoplasm, indicating aberrant proliferation from all three germ layers. This teratoma was only one abnormality in a complex of developmental changes, followed for 28 months, which appeared in a single generation of sibling 2-week-old Xenopus larvae. Two hundred larvae produced by an apparently normal adult pair initially showed ocular defects. including microphthalmia, anophthalmia and tumours projecting near the eyes. During further development up to 28 months, mediastinal tumours developed in nine frogs; these tumours were associated with reduced growth, the frogs reaching only 13-20% of normal weight, and greater enhanced ventral pigmentation. (C) 2000 Harcourt Publishers Ltd.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER VERLAG  

The Ran gene family encodes small GTP binding proteins that are associated with a variety of nuclear processes. We isolated a Xenopus Ran cDNA and analyzed the pattern of expression of this gene during embryogenesis. Ran is expressed maternally and later in the CNS, neural crest, mesenchyme, eyes, and otic vesicles. However, expression is not detected in the somites or the notochord.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

We characterized Xenopus SIP1 (XSIP1), Smad interacting protein, from activin-treated animal caps by differential screening. The XSIP1 is very similar to mouse SIP1 in the protein coding region including the zinc huger domain and homeodomain. The expression pattern was analyzed by RT-PCR and whole mount in situ hybridization. XSIP1 expression was initially restricted to the dorsal marginal zone in the late gastrula and was subsequently expressed at the lateral edge of neural plate and, in the tailbud stage, in the forebrain, neural tube, and eye. Overexpression of XSIP1 at the animal caps resulted in activation of anterior neural markers without mesodermal markers. Ectopic expression of XSIP1 induced enlargement of neural cells and disordered eye formation. In addition to abnormal head phenotypes, many embryos were short-tailed. Our findings suggest that XSIP1 is a transcriptional repressor, which may be involved in the activin-dependent signal pathway, (C) 2000 Academic Press.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：CELLULAR & MOLECULAR BIOLOGY  

Activin uptake into Xenopus oocytes was studied by several complementary methods. Immunocytochemistry of adult ovary localized activin and follistatin in the cytoplasm of vitellogenic oocytes and surrounding follicle cells. Surface plasmon resonance analysis of protein interaction kinetics indicated that while follistatin or a complex of activin-follistatin bound to yolk vitellogenin, activin alone did not. Radioactive tracer analysis measured specific incorporation of activin by viable oocytes in vitro, Together, the results suggest that vitellogenic oocytes can import activins from follicle cells and that follistatin may act as a chaperone for binding activin to vitellogenin in yolk platelets.

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Publishing type：Research paper (scientific journal)  

In most ectotherms, environmental temperature has differential effects on growth and differentiation. For example, amphibian size at maturity decreases with increasing temperature. To address how radiant heat in the form of far-infrared radiation (FIR) may affect development of the aquatic ectotherm Xenopus laevis, we continuously irradiated swimming larvae as they developed into young adults. Here we report evidence that FIR promotes growth of these organisms in an aqueous environment. ©1999 COSPAR. Published by Elsevier Science Ltd.

DOI： 10.1016/S0273-1177(99)00347-6

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Out of a Xenopus neurula cDNA library, we isolated a clone which encodes a 52.4-kDa protein highly similar to the mouse interferon regulatory factor, IRF-6, whose function is unknown. The mRNA of this gene, named xIRF-6, seems to be maternally transmitted, but its amount rapidly decreases after the tailbud stage. Whole-mount in situ hybridization showed that xIRF-6 mRNA is expressed in the presumptive semitic mesoderm in the late gastrula, and then confined to a segment of posterior somite during the neurula through the tailbud stage. The temporally and spatially limited expression of the xIRF-6 gene product may contribute to the transcriptional regulation of specific genes which are necessary for the development of the posterior somites. (C) 1997 Elsevier Science B.V.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL SCIENCE  

Na+, K+-ATPase participates in reabsorption of ions and water and produces an electrochemical gradient between the intra-and extracellular spaces across the cell membrane. It also plays an important role in many developmental phenomena such as a blastocoele formation and neural formation. To elucidate the expression pattern of Na+, K+-ATPase in the Xenopus embryo, the spatial expression patterns of the Na+, K+-ATPase a subunit were studied in a normal embryo by whole-mount in situ hybridization. These transcripts were localized around the dorsal blastopore at the gastrula stage, in the neural tube at the neurula stage, and then in the pronephros and cloaca at the tail-bud stage. To study the function of Na+, K+-ATPase in embryogenesis after mid-blastula transition, the expression of the Nat, K+-ATPase a subunit was inhibited by the injection of specific antisense RNA. Embryos injected with Na+, K+-ATPase antisense RNA showed inhibition of gastrulation. When antisense RNA was injected into the dorsal blastomeres, head differentiation was markedly inhibited. These results suggest that this transcript plays an important role during gastrulation and head differentiation.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIRKHAUSER VERLAG AG  

As a first step towards providing a conceptual approach to understanding similarities and differences in the mechanisms which guide inductive interactions among related organisms (e.g. various amphibia), a set of five principles is offered here. These principles were formulated by analyzing literature examples of classical embryological phenomena and by performing experiments with activin, a peptide growth factor which is currently suspected to play for a role in mesoderm induction. Mechanisms which account, at least in part, for the observed differences between anuran and urodele inductive processes can be derived from these principles.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Warszawa, Krakow, Karol Starmach of Freshwater Biology  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：KARGER  

Activin possesses mesoderm-inducing activity, erythroid-differentiating activity, and follicle-stimulating hormone-releasing activity. The chemical structures of the activin molecule are formed by a combination of two beta-subunit peptides of inhibin. Inhibin is a dimer consisting of an alpha and beta subunit. To examine the mesoderm-inducing activity of these substances, we tested several configurations including : (1) two types of alpha-subunit peptide; (2) two types of inhibin A and B dimer; (3) beta(A)-subunit peptide monomer; (4) three types of activins A, AB and B, and (5) follistatin (activin-binding protein) by the animal cap assay using Xenopus laevis ectoderm, and by the erythroid-differentiating factor (EDF) test. Activins, which are composed of dimeric inhibin beta(A)- or beta(B)-subunit peptides, had the highest mesoderm-inducing and EDF activities. The monomeric beta(A)-subunit peptide exhibited mesoderm-inducing and EDF activities that were much lower than activin A. The inhibitory effect of follistatin on mesodermal induction by the beta(A)-subunit peptide was also lower than that of activin. Both inhibins A and B had very weak mesoderm-inducing activity and no EDF activity. The two types of inhibin (a)lpha-subunit monomer had little mesoderm-inducing activity and no EDF activity. The mesoderm induction caused by activin A was not suppressed by the addition of the alpha-subunit monomer and inhibin. The mesoderm-inducing activity in relation to the chemical structures of the monomeric and/or dimeric inhibin alpha and beta(A) subunits is discussed.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：UNIV BASQUE COUNTRY PRESS  

We reviewed cell differentiation and morphogenesis by mesoderm-inducing factors during amphibian embryogenesis. Recently, two kinds of growth factors, activin and FGF, have been identified as influential candidates for natural mesoderm-inducing factor in amphibian development. These factors are present in early Xenopus embryos. In particular, activin has been shown to induce many kinds of mesodermal tissues in a dose-dependent manner. Activin-treated ectodermal sheet (animal cap) actsasan organizer causing gene expression, mesoderm formation and functional events such as secondary axis formation. Follistatin, an activin-specific binding protein, also present in the early Xenopus embryo, makes a complex with activin. Follistatin protein exerts no inducing activity of Xenopus animal cap. Endogenous follistatin may, however, play the role of an activin regulation factor. Endogenous actions of activin and FGF were studied using injection of their receptor mRNAs. Disruption of the FGF signaling pathway by its non-functional dominant negative receptors produced trunk and tail defects. In the case of activin, an embryo cannot form axial structures. Animal-half blastomeres from the late 8-cell stage Xenopus embryo respond to activin, and there are prepatterns in ventral and dorsal cells from very early stages. The timing of mesoderm induction during development and the relationship between the inducing factors and competent cells are discussed in this report. Differentiation of tissues and organized formation of organs can be understood as a system of serial inductive reactions originating from the organizer. We have attempted to construct a model of organizer formation based on the results of recent studies.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

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Language：Japanese   Publishing type：Research paper (scientific journal)   Publisher：日本生物物理学会  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIRKHAUSER VERLAG AG  

A new cell line (XTY) was derived from a tumor of a female Xenopus laevis. This cell line has been proliferating in standard amphibian culture medium for more than 4 years (470 generations) and has a doubling time of 75.5 h at 25-degrees-C. The cells aggregate into large groups, and their stellate morphology and the expression of desmin demonstrated by immunocytochemistry suggest that their origin is not epithelial.

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ZOOLOGICAL SOC JAPAN  

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Language：Japanese   Publisher：裳華房  

CiNii Books

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Web of Science

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：日本医学館  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：羊土社  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：発達腎研究会  

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Language：Japanese  

CiNii Books

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ACADEMIC PRESS INC  

Web of Science

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

Web of Science

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：裳華房  

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Language：Japanese   Publisher：裳華房  

CiNii Books

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：中山書店  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：東京大学出版会  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：日本医事新報社  

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：日本アイソトープ協会  

DOI： 10.3769/radioisotopes.43.10_649

CiNii Books

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Other Link： http://search.jamas.or.jp/link/ui/1995242299

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (other)   Publisher：秀潤社  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Oral presentation (general)  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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Language：Japanese   Presentation type：Public lecture, seminar, tutorial, course, or other speech  

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