Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier BV  

We previously reported that Tbx6, a T-box transcription factor, is required for the differentiation of ventral body wall muscle and for segment formation and somitic muscle differentiation. Here, we show that Tbx6 is also involved, at later stages, in cartilage differentiation from the cranial neural crest and head muscle development. In Tbx6 knockdown embryos, the cranial neural crest was shown to be correctly induced at the border of the neural plate and migrated in a slightly delayed manner, but finally reached positions in the pharyngeal arches nearly similar to those in the normal embryos as revealed by in situ hybridization and the neural crest-transplantation experiments. However, the neural crest cells failed to maintain Sox9 expression. Tbx6 knockdown also reduced the expression of Tbx1, another T-box gene expressed in more anterior paraxial structures. Tbx1 knockdown caused phenotypes milder but similar to those of Tbx6 morphants, including reduced formation of head muscles and cartilages, and attenuated Sox9 expression. Furthermore, the phenotypes caused by Tbx6 knockdown were partially rescued by Tbx1 plasmid injection. These results suggest that Tbx6 is involved in the cranial cartilage and head muscle development by regulating anterior paraxial genes such as Tbx1 and Sox9.

DOI： 10.1016/j.ydbio.2010.07.028

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

Adipose tissue-derived mesenchymal stem cells (ASCs) have been reported to be multipotent and to differentiate into various cell types, including osteocytes, adipocytes, chondrocytes, and neural cells. Recently, many authors have reported that ASCs are also able to differentiate into vascular endothelial cells (VECs) in vitro. However, these reports included the use of medium containing fetal bovine serum for endothelial differentiation. In the present study, we have developed a novel method for differentiating mouse ASCs into VECs under serum-free conditions. After the differentiation culture, over 80% of the cells expressed vascular endothelial-specific marker proteins and could take up low-density lipoprotein in vitro. This protocol should be helpful in clarifying the mechanisms of ASC differentiation into the VSC lineage.

DOI： 10.1016/j.bbrc.2010.08.029

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

The neural crest (NC) is a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. NC cells differentiate into a vast range of cells, including neural cells, smooth muscle cells, bone and cartilage cells of the maxillofacial region, and odontoblasts. The molecular mechanisms underlying NC induction during early development remain poorly understood. We previously established a defined serum-free culture condition for mouse embryonic stern (mES) cells without feeders. Here, using this defined condition, we have developed a protocol to promote mES cell differentiation into NC cells in an adherent monolayer culture. We found that adding bone morphogenetic protein (BMP)-4 together with fibroblast growth factor (FGF)-2 shifts mES cell differentiation into the NC lineage. Furthermore, we have established a cell line designated as P0-6 that is derived from the blastocysts of P0-Cre/Floxed-EGFP mice expressing EGFP in an NC-lineage-specific manner. P0-6 cells cultured using this protocol expressed EGFP. This protocol could be used to help clarify the mechanisms by which cells differentiate into the NC lineage and to assist the development of applications for clinical therapy.

DOI： 10.1387/ijdb.103173ya

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BENTHAM SCIENCE PUBL LTD  

The glycan-binding profile of a beta-galactoside-binding 15 kDa lectin (Galectin-1) purified from the oocytes of the American bullfrog, Rana catesbeiana, was studied using 61 pyridyl-aminated oligosaccharides by frontal affinity chromatography. Human blood type-A-hexasaccharide (GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-4GlcNAc beta 1-4Gal beta 1-4Glc) was found to exhibit the strongest ligand binding to the galectin while Forssman antigen (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc) and type-A-tetrasaccharide (GalNAc alpha 1-3(Fuc alpha 1-2)Gal beta 1-4GlcNAc beta 1-4Glc) were also extensively recognized. The kinetics of affinity of galectin-1 to type-A oligosaccharide was analysed by surface plasmon resonance using neoglycoprotein with type-A oligosaccharides. R. catesbeiana oocyte galectin adhered to human rhabdomyosarcoma cells dose dependently and the activity was specifically cancelled by the neoglycoprotein. It was concluded that galectin-1 from R. catesbeiana oocytes possesses different and rare glycan-binding properties from typical members in galectin family.

DOI： 10.2174/092986609788490104

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Language：Japanese   Publisher：日本組織培養学会  

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

Bowline, which is a member of the Xenopus Bowline/Ripply family of proteins, represses the transcription of somitogenesis-related genes before somite segmentation, which makes Bowline indispensable for somitogenesis. Although there are three bowline/Ripplyfamily genes in each vertebrate species, it is not known whether the Bowline/Ripply family proteins share a common role in development. To elucidate their developmental roles, we examined the expression patterns and functions of the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline, and a novel member of this protein family, xRipply3. We found that the expression patterns of bowline and ledgerline overlapped in the presomitic mesoderm (PSM), whereas ledgerline was additionally expressed in the newly formed somites. In addition, we isolated xRipply3, which is expressed in the pharyngeal region. Co-immunoprecipitation assays revealed that Ledgerline and xRipply3 interacted with T-box proteins and the transcriptional co-repressor Groucho/TLE. In luciferase assays, xRipply3 weakly suppressed the transcriptional activity of Tbx1, while Ledgerline strongly suppressed that of Tbx6. In line with the repressive role of Ledgerline, knockdown of Ledgerline resulted in enlargement of expression regions of the somitogenesis-related-genes mespb and Tbx6. Inhibition of histone deacetylase activity increased the expression of mespb, as seen in the Bowline and Ledgerline knockdown experiments. These results suggest that the Groucho-HDAC complex is required for the repressive activity of Bowline/Ripply family proteins during Xenopus somitogenesis. We conclude that although the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline and xRipply3 are expressed differentially, they all act as negative regulators of T-box proteins.

DOI： 10.1387/ijdb.082823kh

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T-box transcription factor tbx6 and basic-helix-loop-helix transcription factor pMesogenin1 are reported to be involved in paraxial mesodermal differentiation. To clarify the relationship between these genes in Xenopus laevis, we isolated pMesogenin2, which showed high homology with pMesogenin1. Both pMesogenin1 and 2 appeared to be transcriptional activators and were induced by a hormone-inducible version of Xtbx6 without secondary protein synthesis in animal cap assays. The pMesogenin2 promoter contained three potential T-box binding sites with which Xtbx6 protein was shown to interact, and a reporter gene construct containing these sites was activated by Xtbx6. Xtbx6 knockdown reduced pMesogenin1 and 2 expressions, but not vice versa. Xtbx6 and pMesogenin1 and 2 knockdowns caused similar phenotypic abnormalities including somite malformation and ventral body wall muscle hypoplasia, suggesting that Xtbx6 is a direct regulator of pMesogenin1 and 2, which are both involved in somitogenesis and myogenesis including that of body wall muscle in Xenopus laevis.

DOI： 10.1002/dvdy.21791

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

During vertebrate somitogenesis, various transcriptional factors function coordinately to determine the position of the somite boundary. Previously, we reported on the signaling crosstalk that occurs between two major transcription factors involved in somitogenesis, Tbx6 and mespb/mesp2. These factors synergistically activated the expression of a downstream gene, bowline/Ripply2, which is essential for precise formation of the somite boundary. However, the molecular mechanism underlying this synergistic effect remains unclear. In this report, we found that the Tbx6 and mespb proteins interacted physically with each other. Pulldown assays with various deletion mutants of these proteins identified the essential domains for this physical interaction. Finally, we found that interference with the physical interaction by a dominant-negative form of mespb, mespb Delta DBD, abrogated the expression of the bowline gene during Xenopus somitogenesis. These results indicate that the appropriate expression of bowline/Ripply2 is regulated by a direct interaction between the Tbx6 and mespb proteins during Xenopus somitogenesis. (C) 2008 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2008.05.083

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

T-box factor, Tbx6, is a prerequisite for somite segmentation in vertebrates. We recently identified a negative regulator of Tbx6, Bowline, which represses the expression of genes involved in somite segmentation by suppressing the transcriptional activity of Tbx6. According to this function, bowline gene expression is restricted to the most anterior presomitic mesoderm where the somite segmentation program terminates, although it remains unclear how bowline expression is activated. To address this, we investigated the cis-regulatory region of bowline. Measuring luciferase activity driven by the bowline promoter, we found that Tbx6, Thylacine1, and E47 synergistically activate bowline expression in vitro. We also found that Tbx6, Thylacine1, and E47 are spatiotemporally sufficient to induce bowline expression in Xenopus somitogenesis. Our findings indicated that besides being a negative regulator of Tbx6, bowline itself is also regulated by Tbx6, suggesting the negative feedback loop of Tbx6-Bowline in the termination step of somite segmentation. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2007.10.015

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)  

T-box proteins are important transcriptional regulators in animal development. We searched the Xenopus laevis expressed sequence tag (EST) database using zebrafish tbx24 (Ztbx24) as a query and found a sequence. We then obtained corresponding clones from a neurula cDNA library. This novel gene has a T-box showing 53% homology with Xenopus laevis tbx6 (Xtbx6) and 51% with Ztbx24 at the amino acid level and is relatively close to Xtbx6 indicated by alignment analysis. In situ hybridization showed that it is expressed in the paraxial mesoderm in the caudal region in a very similar manner to Xtbx6, with a slight difference in that the former is emphasized more dorsally and has a more restricted distribution along the antero-posterior axis. From these results we named this gene Xtbx6r (tbx6-related). Xtbx6r or Xtbx6r-EnR, when overexpressed in animal caps, induced anterior neural markers but not mesodermal markers. In contrast, Xtbx6r-VP16, Xtbx6 and Ztbx24 induced various mesodermal markers. These results indicate that Xtbx6r is a transcriptional repressor and has activity different from that of Xtbx6 or Ztbx24. Xtbx6r induced Otx2, XAG and Pax6 in animal caps. This activity differed from that of Xbra-EnR or Xtbx6-EnR, suggesting that some differences in biological activity exist among the tested repressor-type T-box genes. Depletion of Xtbx6r by antisense morpholino oligo produced curved embryos, but did not affect expressions of MyoD, Myf5, XWnt8 or thylacine2, nor inhibited muscle differentiation or segmentation. The results of knockdown and overexpression experiments suggest that Xtbx6r is involved in some morphogenesis in the paraxial mesoderm.

DOI： 10.1387/ijdb.062177sy

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Authorship：Last author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING  

Essential roles for GATA factors in the development of endoderm have been reported in various animals. A Drosophila GATA factor gene, serpent (srp, dGATAb, ABF), is expressed in the prospective endoderm, and loss of srp activity causes transformation of the prospective endoderm into ectodermal foregut and hindgut, indicating that srp acts as a selector gene to specify the developmental fate of the endoderm. While srp is expressed in the endoderm only during early stages, it activates a subsequent GATA factor gene, dGATAe, and the latter continues to be expressed specifically in the endoderm throughout life. dGATAe activates various functional genes in the differentiated endodermal midgut. An analogous mode of regulation has been reported in Caenorhabditis elegans, in which a pair of GATA genes, end-1/3, specifies endodermal fate, and a downstream pair of GATA genes, elt-2/7, activates genes in the differentiated endoderm. Functional homology of GATA genes in nature is apparently extendable to vertebrates, because endodermal GATA genes of C. elegans and Drosophila induce endoderm development in Xenopus ectoderm. These findings strongly imply evolutionary conservation of the roles of GATA factors in the endoderm across the protostomes and the deuterostomes.

DOI： 10.1111/j.1440-169X.2005.00836.x

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Regional differentiation along the dorsoventral (DV) axis of the Drosophila embryo primarily depends on a graded BMP signaling activity generated by Decapentaplegic (Dpp) and Screw (Scw). We have identified triplicated Dpp and Scw target genes Dorsocross1, 2 and 3 (Doc1, 2, 3) that have a conserved T-box domain related to the vertebrate Tbx6 subfamily and act redundantly to induce dorsal structures. Doc genes are expressed in the dorsal region in the early blastoderm. After gastrulation, newly expressed Doc appears in a segmental pattern in the ectoderm. This expression correlates spatially with the second phase of Dpp expression in the ectoderm. Doc expression in the early blastoderm is abolished in either dpp or scw mutant embryos, whereas the ectodermal segmented expression depends only on Dpp. Inactivation of Doc genes with RNAi dramatically affected the development of amnioserosa and wing disc primordia, both of which depend on high levels of BMP signaling, although leg disc primordium, which depends on low levels of BMP, remained intact. Doc1 mRNA expressed in Xenopus embryos induced ventral mesoderm, suppressed activin-induced events and induced Xvent genes, which are analogous to the effects of native Tbx6 and its upstream regulator, BMP-4. These results suggest that the Tbx6 subfamily act in the BMP signaling pathway required for embryonic patterning in both animals.

DOI： 10.1016/j.ydbio.2003.09.034

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DOI： 10.1046/j.1440-169X.2002.044001095.x

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Japanese Society of Developmental Biologists  

Tbx6 is a member of the T-box gene family. Studies of knockout mice indicate that Tbx6 is involved in somite differentiation. In the present study, we cloned Tbx6 from another vertebrate species, namely Xenopus laevis, and studied its roles in development. The expression of Tbx6 in Xenopus started from the early gastrula stage, reached a peak during the late gastrula to neurula stages and then declined. Initial expression of Tbx6 was observed in the paraxial mesoderm during the gastrula stage. The Tbx6-expressing region spread anteriorly and ventrally in the neurula stage. In the tailbud stage, the area of expression shrank caudally and was finally restricted to the tip of the tailbud. Overexpression of Tbx6 mRNA in dorsal blastomeres caused atrophy of the neural tube and inhibited differentiation of the notochord. Animal cap explants overexpressing Tbx6 or Tbx6VP16 mRNA, but not Tbx6EnR mRNA, differentiated mainly into ventral mesodermal tissues. This suggests that Tbx6 is a transcriptional activator. Higher doses of Tbx6 or Tbx6VP16 mRNA caused hardly any muscular differentiation. However, coinjection of Tbx6 mRNA with noggin mRNA elicited marked muscle differentiation. These results suggest that Tbx6 is implicated in ventral mesoderm specification but is involved in muscle differentiation when acting together with the dorsalizing factor noggin.

DOI： 10.1046/j.1440-169X.2001.00606.x

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

The effects of activin A and follistatin on the release of follicle-stimulating hormone (FSH), luteinizing hormone (LH), growth hormone (GH) and prolactin (PRL) from dispersed pituitary cells of the bullfrogs Rana catesbeiana were studied. Activin A stimulated the release of FSH, GH, and PRL dose-dependently, but not that of LH. Follistatin suppressed the activin-induced FSH, GH, and PRL release, but did not affect the basal secretion of those hormones. From the results obtained in this experiment, together with the previously obtained findings that activin B enhanced the release of FSH, LH, GH, and PRL, we conclude that activin A, in addition to activin B, influences the function of multiple types of pituitary cells in the bullfrog.

DOI： 10.2108/zsj.17.971

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

Occurrence of immunoreactive activin/inhibin beta(B) in the bullfrog (Rana catesbeiana) pituitary was investigated immunocytochemically by use of antibody against Xenopus activin/inhibin beta(B) subunit. Thyrotropes were demonstrated to contain activin/inhibin beta(B)-immunoreactive substances. Moreover, immunoelectron microscopy revealed that in the secretory granules of thyrotropes and, to a lesser extent, in those of gonadotropes, activin/inhibin beta(B)-immunoreactive substances were present. Based on this observation, we investigated the effect of activin B on the release of gonadotropins from dispersed anterior pituitary cells of the bullfrog. Activin B stimulated the release of not only follicle-stimulating hormone (FSH) but also luteinizing hormone (LH) dose dependently. Under the culture conditions used in this experiment, inhibin B, as well as follistatin, did not affect the basal levels of LH and FSH, but they suppressed the activin-induced release of these hormones. This is the first study on the effect of activin on pituitary hormone secretion in lower tetrapods. (C) 2000 Academic Press.

DOI： 10.1006/gcen.2000.7456

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Proceedings of the National Academy of Sciences  

In early development of Xenopus laevis, it is known that activities of polypeptide growth factors are negatively regulated by their binding proteins. In this study, follistatin, originally known as an activin-binding protein, was shown to inhibit all aspects of bone morphogenetic protein (BMP) activity in early Xenopus embryos. Furthermore, using a surface plasmon resonance biosensor, we demonstrated that follistatin can directly interact with multiple BMPs at significantly high affinities. Interestingly, follistatin was found to be noncompetitive with the BMP receptor for ligand binding and to form a trimeric complex with BMP and its receptor. The results suggest that follistatin acts as an organizer factor in early amphibian embryogenesis by inhibiting BMP activities by a different mechanism from that used by chordin and noggin.

DOI： 10.1073/pnas.95.16.9337

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Galectins are a family of lectins that recognize beta-D-galactosides independently of calcium ions, and are widely distributed in animals, To characterize a galectin previously purified from oocytes of Rana catesbeiana (American bullfrog), we studied its distribution and localization in several tissues from this frog, Hemagglutination assay and western blotting showed that this lectin is present in many tissues including the liver, skin, kidney, skeletal muscle, and sciatic nerve, but is particularly concentrated in the ovary, Light microscopic immunohistochemistry showed that this lectin is localized in such places as cell-cell junctions, basement membranes, extracellular matrix, or secretory substances in several organs, indicating that this galectin is mainly distributed extracellularly, However, in the ovary, light microscopy showed that this lectin is present in or associated with the yolk platelet, Electron microscopy further revealed that it is localized in the periphery of the yolk platelet (the yolk plasm), but not in the cortical granule, These results indicate that Rana oocytes contain abundant galectin in their yolk platelets in contrast to Xenopus laevis oocytes, which have been found not to contain galectins but other classes of lectins in their yolk platelets and cortical granules.

DOI： 10.1093/glycob/7.8.1159

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

Many chemicals released into the environment have estrogenic activity and can disrupt animal development and the function of endocrine systems. In order to study the effects of estrogens on aquatic animals, we examined the effects of certain estrogens on early development in Xenopus laevis. X. laevis embryos were kept in water containing 10(-10), 10(-9), 10(-7), 10(-6), and 10(-5) M 17 beta-estradiol (E-2); 17 alpha-estradiol; diethylstilbestrol (DES); 10(-5) M progesterone (P); or dihydrotestosterone (DHT) beginning at developmental stage 3. Survival rates of the embryos developed in water containing 10(-10)-10(-6) M E-2 or DES, all concentrations of 17 alpha-estradiol, and 10(-5) M P or DHT, which were over 70% after stage 48, whereas the rates of the embryos treated with 10(-5) M E-2 and DES decreased remarkably after stage 27 and all embryos were dead by stages 42 and 32, respectively. Embryos treated with 10(-5) M E-2 showed malformations of the head and abdomen and suppressed organogenesis, including crooked vertebrae at stage 38; the head was smaller and the abdomen was larger than in the controls. Similar effects were observed in embryos developed in 10(-5) M DES but not in 10(-5) M 17 alpha-estradiol, P, or DHT. After 10(-5) M E-2 treatment, abnormalities were induced only when the treatment was started before stage 39. However, on day 30 after fertilization, the stage of the embryos treated with 10(-6) M E-2 was more progressed than that of the controls. Estrogen receptor (ER 4) mRNA was examined in eggs, embryos, and adult female liver by reverse-transcription polymerase chain reaction. ER4 mRNA was expressed in adult liver, unfertilized and fertilized eggs, and embryos, but ER3 mRNA was not expressed. ER4 mRNA in 10(-6) and 10(-5) M E-2-treated embryos showed different expression patterns, which may result from the diverse developmental effects of E-2. The present results demonstrate that 10(-5) M E-2 and DES induced embryo death and malformations and that ER may be involved in the induction of various developmental defects in X. laevis embryos. (C) 1997 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-010X(19970701)278:4<221::AID-JEZ3>3.0.CO;2-R

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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

An antibody against the Xenopus activin/inhibin beta(B) subunit (94-107) was raised in a rabbit. Using this antibody, the distribution of activin/inhibin beta(B) immunoreactivity in the pituitary of adult X. laevis was studied. beta(B) immunoreactivity was detected in gonadotrophs, thyrotrophs, and somatotrophs under light microscopy. Electron microscopy revealed that a beta(B)-immunoreactive substance exists in LH, TSH, and GH granules, in contrast to findings in the rat and goldfish. These results indicate that the expression of activin/inhibin beta(B) in pituitary cells is not consistent among vertebrate species. (C) 1996 Academic Press, Inc.

DOI： 10.1006/gcen.1996.0039

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ZOOLOGICAL SOC JAPAN  

Neural differentiation in amphibian embryos had been thought to start after gastrulation because of vertical neural induction from the underlying dorsal involuting marginal zone (DIMZ). However, recent studies show that another mode of induction, namely the planar induction, also directs neural differentiation in Xenopus laevis. Here, we attempted to specify when the planar induction occurs. From middle blastula to early gastrula stages, explants that included animal cap (AC) and dorsal noninvoluting marginal zone (DNIMZ), but not DIMZ were prepared. Neural differentiation was detected in such explants (named AC-DNIMZ explants) from the early gastrula (St. 10+) but not from the late blastula (St. 9) or earlier embryos, indicating that the planar induction occurs at about St. 10+ To assess the further effect of planar induction on neural differentiation, Keller sandwiches were prepared from St. 10+ embryos, and cultured for 2 similar to 6 hr in vitro, after which the explants were separated into AC-DNIMZ and DIMZ parts. AC-DNIMZ part differentiated a little larger neural tissues, suggesting that the planar neural induction during the gastrula stages promote neural differentiation. Neural tissues in these explants could be detected by histology and immunohistochemistry using a monoclonal antibody specific for neural tissues, as opposed to earlier studies that indicated the planar induction to be insufficent for inducing morphological neural structures.

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The Xenopus homologue of sonic hedgehog (Xhh) was detected in Xenopus embryos at stages 13 and 31 by RT-PCR, but it was not expressed in explants isolated from the animal hemisphere of Xenopus embryos at stage 8-9. Treatment of the animal cap with activin (1-100 ng/ml) induced the expression of Xhh. However, it was not induced by 100 ng/ml basic fibroblast growth factor (bFGF). Whole mount in situ hybridization confirmed the expression of Xhh in the animal cap treated with activin. The expression of Xhh induced by activin was not inhibited in the presence of cycloheximide, suggesting that Xhh is an early response gene induced by activin. (C) 1995 Academic Press, Inc.

DOI： 10.1006/bbrc.1995.1144

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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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Authorship：Lead author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

We found a binding protein for activin and follistatin in serum from female Xenopus laevis and identified it as vitellogenin, which is synthesized in the liver and transported into yolk platelets. Then, we investigated the localization of activin and follistatin proteins in early Xenopus oocytes (stage 6) by electron microscopic immunolabeling with gold colloidal particles. The protein molecules were found to be localized uniformly in oocyte yolk platelets, but not in other cytoplasmic organelles. These findings suggest a novel role of yolk platelets as a reservoir for inductive signals transported by vitellogenin in the differentiation and patterning of cells in Xenopus embryos. (C) 1994 Academic Press, Inc.

DOI： 10.1006/bbrc.1994.1954

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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 isoforms 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. © 1994 by Academic Press, Inc.

DOI： 10.1006/dbio.1994.1143

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Xenopus follistatin and activins were purified from a Xenopus laevis cell line (XTC-F1) by four purification steps consisting of consecutive affinity chromatography on dextran sulfate-Sepharose and Sulfate Cellulofine, fast protein liquid chromatography gel permeation, and reverse-phase high performance liquid chromatography (HPLC). Our results thus obtained indicated that almost equimolar amounts of activins A, AB, and B were found to be present as a complex with follistatin (activin-binding protein) in the conditioned medium of XTC-F1 cells. Reverse-phase HPLC of the complex gave Xenopus follistatin and activins A, AB, and B. The purified Xenopus follistatin showed four major bands in a molecular mass range from 34 to 39 kDa by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. The ability of each form of the protein to specifically bind activin was determined by activin-binding assay and ligand blotting analysis. Each protein was found to have the same NH2-terminus and its sequence was very homologous to that of mammalian follistatin. Several criteria including immunoblotting analysis and various functional assays revealed the existence of three isoforms of activins A, AB, and B in Xenopus, as in mammals. Xenopus activins significantly induced both ventral and dorsal mesoderm in explants of Xenopus blastula cells that would otherwise form epidermis. In a dose-dependent manner of each isoform of activin, the induced explants were able to differentiate into blood-like cells, coelomic epithelium, mesenchyme, muscle, and notochord. The induction patterns of three Xenopus activins were essentially the same. The mesoderm induction by the purified Xenopus activins was shown to be inhibited stoichiometrically by the purified Xenopus follistatin. These results indicate that Xenopus XTC-F1 cells secrete several molecular forms of follistatin/activin-binding protein and three isoforms of activins AB and B in addition to activin A. © 1993 by Academic Press, Inc.

DOI： 10.1006/dbio.1993.1227

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DOI： 10.1111/j.1440-169X.1993.00123.x

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

To examine whether activin binds to follistatin, an activin-binding protein, to form a complex in vivo, we attempted to purify activin-follistatin complex from porcine follicular fluid. Our results thus obtained indicated that almost equimolar amounts of activins A, AB, and B are present as a complex with follistatin in the follicular fluid. Reverse-phase high performance liquid chromatography of the purified complex yielded follistatin and activins A, AB, and B. The activity of the purified activin B was found to be significantly lower than those of other activins in various assay systems such as stimulation of follicle-stimulating hormone secretion, induction of erythrodifferentiation, and potentiation of expression of gonadotropin receptors on ovarian cells. Moreover, binding of I-125-activin A to erythroleukemic cells which are activin-responsive was competed by activin B with approximately 10-fold lower potency compared with other activins. In contrast to these results, activin B was proved to have a potent Xenopus mesoderm-inducing activity, comparable with that of other activins. This indicates that, unlike activins A and AB, activin B can only elicit mesoderm-inducing activity and cannot function in other biological systems, suggesting a specific role of activin B in early development and unknown biological functions.

DOI： 10.1016/s0021-9258(18)42014-5

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

DOI： 10.1016/0006-291X(92)91199-Z

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Activin A, a member of the transforming growth factor-beta superfamily, has recently been found to have potent mesoderm-inducing activity on isolated early Xenopus animal-cap cells. We measured the activin activity of the Xenopus egg extract by using an erythroid-differentiating test with Friend leukemia cells. The results showed that an activin homologue is, indeed, contained in unfertilized eggs and blastulae of Xenopus laevis in a considerable amount. This activity was eluted at the same retention time as human activin A when fractionated by reversed-phase HPLC. Furthermore, the fraction containing erythroid-differentiating factor activity had mesoderm-inducing activity on Xenopus animal-cap cells. The mesoderm-inducing activity of this fraction was suppressed when coincubated with follistatin, an activin-binding protein. These results suggest that an endogenous activin may be a natural mesoderm-inducing factor acting in Xenopus embryogenesis.

DOI： 10.1073/pnas.88.15.6511

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We examined the quality of mesoderm induced by the action of activin A on the Xenopus presumptive ectoderm when various concentrations and treatment times were employed. The minimum concentration of activin A to induce mesodermal tissues was inversely proportional to its treatment time. The explants differentiated into different types of mesodermal tissues, from ventral-type to dorsal-type depending on the concentration of activin A and its treatment time. To confirm whether activin A has a role in establishing axial organization, activin A was injected into the blastocoel of late blastulae. About 70% of the injected embryos formed secondary tail-shaped outgrowths in which muscle and neural tube differentiated. The amount of activin A to form secondary outgrowths was 0.5-2.5 pg, roughly consistent with the amount estimated from in vitro experiments. As we have detected almost the same amount of activin homologue in the early embryos (Asashima et al., 1991 a), we speculate that activin A may be the natural mesodermal inducer, and that it is responsible for establishing axial organization in the Xenopus embryo.

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

Human recombinant activin A, which is identical with erythroid differentiation factor (EDF), was tested for its mesoderm-inducing activity in concentrations from 0.3-50 ng/ml, using ectoderm of Xenopus late blastula (Stage 9) as the responding tissue. At a low concentration of activin A, blood-like cells, mesenchyme, and coelomic epithelium were induced; at a moderate concentration muscle and neural tissue, and at a high concentration notochord. Activin A thus induced all mesodermal tissues in a dose-dependent manner, such that a low dose induced ventral structures and a high dose induced dorsal structures. Activin may act as an intrinsic inducing molecule responsible for establishing the dorso-ventral axis in early Xenopus development.

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

The induction of mesoderm is an important process in early amphibian development. In recent studies, activin has become an effective candidate for a natural mesoderm-inducing factor. In the present study, we show that follistatin, an activin-binding protein purified from porcine ovary, inhibits the mesoderm-inducing activity of recombinant human activin A (rh activin A), which is identical to the erythroid differentiation factor (EDF). The quantity of follistatin required for effective suppression of activin was more than three-fold that of activin (w:w). Follistatin also inhibited the mesoderm-inducing activity of the vegetalizing factor purified from chick embryos, suggesting that the vegetalizing factor is closely related to activin.

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

The erythroid differentiation capacity of the HPLC-purified mesoderm- and endoderm-inducing vegetalizing factor from chicken embryos and of recombinant erythroid differentiation factor (EDF = activin A), an evolutionary highly conserved member of the TGF-beta protein superfamily have been compared. Both factors stimulate the synthesis of hemoglobin in erythroleukemia cells in the same concentration range. The EDF-activity of the mesoderm-inducing HPLC-fractions is inhibited by follistatin, an EDF-binding protein. The factor induces in ectoderm of Triturus taeniatus all kinds of mesodermal organs. The wide spectrum of organs is very likely to be induced by secondary interactions. At higher concentration (15 ng/ml), notochord- and endoderm-like tissues are induced in a high percentage.

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

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

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T-box factor, Tbx6, is a prerequisite for somite segmentation in vertebrates. We recently identified a negative regulator of Tbx6, Bowline, which represses the expression of genes involved in somite segmentation by suppressing the transcriptional activity of Tbx6. According to this function, bowline gene expression is restricted to the most anterior presomitic mesoderm where the somite segmentation program terminates, although it remains unclear how bowline expression is activated. To address this, we investigated the cis-regulatory region of bowline. Measuring luciferase activity driven by the bowline promoter, we found that Tbx6, Thylacine1, and E47 synergistically activate bowline expression in vitro. We also found that Tbx6, Thylacine1, and E47 are spatiotemporally sufficient to induce bowline expression in Xenopus somitogenesis. Our findings indicated that besides being a negative regulator of Tbx6, bowline itself is also regulated by Tbx6, suggesting the negative feedback loop of Tbx6-Bowline in the termination step of somite segmentation. (C) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2007.10.015

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DOI： 10.1016/j.ydbio.2003.09.034

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Tbx6 is a member of the T-box gene family. Studies of knockout mice indicate that Tbx6 is involved in somite differentiation. In the present study, we cloned Tbx6 from another vertebrate species, namely Xenopus laevis, and studied its roles in development. The expression of Tbx6 in Xenopus started from the early gastrula stage, reached a peak during the late gastrula to neurula stages and then declined. Initial expression of Tbx6 was observed in the paraxial mesoderm during the gastrula stage. The Tbx6-expressing region spread anteriorly and ventrally in the neurula stage. In the tailbud stage, the area of expression shrank caudally and was finally restricted to the tip of the tailbud. Overexpression of Tbx6 mRNA in dorsal blastomeres caused atrophy of the neural tube and inhibited differentiation of the notochord. Animal cap explants overexpressing Tbx6 or Tbx6VP16 mRNA, but not Tbx6EnR mRNA, differentiated mainly into ventral mesodermal tissues. This suggests that Tbx6 is a transcriptional activator. Higher doses of Tbx6 or Tbx6VP16 mRNA caused hardly any muscular differentiation. However, coinjection of Tbx6 mRNA with noggin mRNA elicited marked muscle differentiation. These results suggest that Tbx6 is implicated in ventral mesoderm specification but is involved in muscle differentiation when acting together with the dorsalizing factor noggin.

DOI： 10.1046/j.1440-169X.2001.00606.x

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

The effects of activin A and follistatin on the release of follicle-stimulating hormone (FSH), luteinizing hormone (LH), growth hormone (GH) and prolactin (PRL) from dispersed pituitary cells of the bullfrogs Rana catesbeiana were studied. Activin A stimulated the release of FSH, GH, and PRL dose-dependently, but not that of LH. Follistatin suppressed the activin-induced FSH, GH, and PRL release, but did not affect the basal secretion of those hormones. From the results obtained in this experiment, together with the previously obtained findings that activin B enhanced the release of FSH, LH, GH, and PRL, we conclude that activin A, in addition to activin B, influences the function of multiple types of pituitary cells in the bullfrog.

DOI： 10.2108/zsj.17.971

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Language：Japanese   Publisher：日本発生生物学会  

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Occurrence of immunoreactive activin/inhibin beta(B) in the bullfrog (Rana catesbeiana) pituitary was investigated immunocytochemically by use of antibody against Xenopus activin/inhibin beta(B) subunit. Thyrotropes were demonstrated to contain activin/inhibin beta(B)-immunoreactive substances. Moreover, immunoelectron microscopy revealed that in the secretory granules of thyrotropes and, to a lesser extent, in those of gonadotropes, activin/inhibin beta(B)-immunoreactive substances were present. Based on this observation, we investigated the effect of activin B on the release of gonadotropins from dispersed anterior pituitary cells of the bullfrog. Activin B stimulated the release of not only follicle-stimulating hormone (FSH) but also luteinizing hormone (LH) dose dependently. Under the culture conditions used in this experiment, inhibin B, as well as follistatin, did not affect the basal levels of LH and FSH, but they suppressed the activin-induced release of these hormones. This is the first study on the effect of activin on pituitary hormone secretion in lower tetrapods. (C) 2000 Academic Press.

DOI： 10.1006/gcen.2000.7456

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Language：Japanese   Publisher：横浜市立大学学術研究会  

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Language：English   Publisher：NATL ACAD SCIENCES  

In early development of Xenopus laevis, it is known that activities of polypeptide growth factors are negatively regulated by their binding proteins, In this study, follistatin, originally known as an activin-binding protein, was shown to inhibit all aspects of bone morphogenetic protein (BMP) activity in early Xenopus embryos. Furthermore, using a surface plasmon resonance biosensor, we demonstrated that follistatin can directly interact with multiple BMPs at significantly high affinities. Interestingly, follistatin was found to be noncompetitive with the BMP receptor for ligand binding and to form a trimeric complex with BMP and its receptor. The results suggest that follistatin acts as an organizer factor in early amphibian embryogenesis by inhibiting BMP activities by a different mechanism from that used by chordin and noggin.

DOI： 10.1073/pnas.95.16.9337

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Language：English   Publisher：OXFORD UNIV PRESS  

Galectins are a family of lectins that recognize beta-D-galactosides independently of calcium ions, and are widely distributed in animals, To characterize a galectin previously purified from oocytes of Rana catesbeiana (American bullfrog), we studied its distribution and localization in several tissues from this frog, Hemagglutination assay and western blotting showed that this lectin is present in many tissues including the liver, skin, kidney, skeletal muscle, and sciatic nerve, but is particularly concentrated in the ovary, Light microscopic immunohistochemistry showed that this lectin is localized in such places as cell-cell junctions, basement membranes, extracellular matrix, or secretory substances in several organs, indicating that this galectin is mainly distributed extracellularly, However, in the ovary, light microscopy showed that this lectin is present in or associated with the yolk platelet, Electron microscopy further revealed that it is localized in the periphery of the yolk platelet (the yolk plasm), but not in the cortical granule, These results indicate that Rana oocytes contain abundant galectin in their yolk platelets in contrast to Xenopus laevis oocytes, which have been found not to contain galectins but other classes of lectins in their yolk platelets and cortical granules.

DOI： 10.1093/glycob/7.8.1159

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Language：English   Publisher：WILEY-LISS  

Many chemicals released into the environment have estrogenic activity and can disrupt animal development and the function of endocrine systems. In order to study the effects of estrogens on aquatic animals, we examined the effects of certain estrogens on early development in Xenopus laevis. X. laevis embryos were kept in water containing 10(-10), 10(-9), 10(-7), 10(-6), and 10(-5) M 17 beta-estradiol (E-2); 17 alpha-estradiol; diethylstilbestrol (DES); 10(-5) M progesterone (P); or dihydrotestosterone (DHT) beginning at developmental stage 3. Survival rates of the embryos developed in water containing 10(-10)-10(-6) M E-2 or DES, all concentrations of 17 alpha-estradiol, and 10(-5) M P or DHT, which were over 70% after stage 48, whereas the rates of the embryos treated with 10(-5) M E-2 and DES decreased remarkably after stage 27 and all embryos were dead by stages 42 and 32, respectively. Embryos treated with 10(-5) M E-2 showed malformations of the head and abdomen and suppressed organogenesis, including crooked vertebrae at stage 38; the head was smaller and the abdomen was larger than in the controls. Similar effects were observed in embryos developed in 10(-5) M DES but not in 10(-5) M 17 alpha-estradiol, P, or DHT. After 10(-5) M E-2 treatment, abnormalities were induced only when the treatment was started before stage 39. However, on day 30 after fertilization, the stage of the embryos treated with 10(-6) M E-2 was more progressed than that of the controls. Estrogen receptor (ER 4) mRNA was examined in eggs, embryos, and adult female liver by reverse-transcription polymerase chain reaction. ER4 mRNA was expressed in adult liver, unfertilized and fertilized eggs, and embryos, but ER3 mRNA was not expressed. ER4 mRNA in 10(-6) and 10(-5) M E-2-treated embryos showed different expression patterns, which may result from the diverse developmental effects of E-2. The present results demonstrate that 10(-5) M E-2 and DES induced embryo death and malformations and that ER may be involved in the induction of various developmental defects in X. laevis embryos. (C) 1997 Wiley-Liss, Inc.

DOI： 10.1002/(SICI)1097-010X(19970701)278:4<221::AID-JEZ3>3.0.CO;2-R

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Language：English   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

An antibody against the Xenopus activin/inhibin beta(B) subunit (94-107) was raised in a rabbit. Using this antibody, the distribution of activin/inhibin beta(B) immunoreactivity in the pituitary of adult X. laevis was studied. beta(B) immunoreactivity was detected in gonadotrophs, thyrotrophs, and somatotrophs under light microscopy. Electron microscopy revealed that a beta(B)-immunoreactive substance exists in LH, TSH, and GH granules, in contrast to findings in the rat and goldfish. These results indicate that the expression of activin/inhibin beta(B) in pituitary cells is not consistent among vertebrate species. (C) 1996 Academic Press, Inc.

DOI： 10.1006/gcen.1996.0039

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Language：English   Publisher：ZOOLOGICAL SOC JAPAN  

Neural differentiation in amphibian embryos had been thought to start after gastrulation because of vertical neural induction from the underlying dorsal involuting marginal zone (DIMZ). However, recent studies show that another mode of induction, namely the planar induction, also directs neural differentiation in Xenopus laevis. Here, we attempted to specify when the planar induction occurs. From middle blastula to early gastrula stages, explants that included animal cap (AC) and dorsal noninvoluting marginal zone (DNIMZ), but not DIMZ were prepared. Neural differentiation was detected in such explants (named AC-DNIMZ explants) from the early gastrula (St. 10+) but not from the late blastula (St. 9) or earlier embryos, indicating that the planar induction occurs at about St. 10+ To assess the further effect of planar induction on neural differentiation, Keller sandwiches were prepared from St. 10+ embryos, and cultured for 2 similar to 6 hr in vitro, after which the explants were separated into AC-DNIMZ and DIMZ parts. AC-DNIMZ part differentiated a little larger neural tissues, suggesting that the planar neural induction during the gastrula stages promote neural differentiation. Neural tissues in these explants could be detected by histology and immunohistochemistry using a monoclonal antibody specific for neural tissues, as opposed to earlier studies that indicated the planar induction to be insufficent for inducing morphological neural structures.

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Language：English   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

The Xenopus homologue of sonic hedgehog (Xhh) was detected in Xenopus embryos at stages 13 and 31 by RT-PCR, but it was not expressed in explants isolated from the animal hemisphere of Xenopus embryos at stage 8-9. Treatment of the animal cap with activin (1-100 ng/ml) induced the expression of Xhh. However, it was not induced by 100 ng/ml basic fibroblast growth factor (bFGF). Whole mount in situ hybridization confirmed the expression of Xhh in the animal cap treated with activin. The expression of Xhh induced by activin was not inhibited in the presence of cycloheximide, suggesting that Xhh is an early response gene induced by activin. (C) 1995 Academic Press, Inc.

DOI： 10.1006/bbrc.1995.1144

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Language：English   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   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

We found a binding protein for activin and follistatin in serum from female Xenopus laevis and identified it as vitellogenin, which is synthesized in the liver and transported into yolk platelets. Then, we investigated the localization of activin and follistatin proteins in early Xenopus oocytes (stage 6) by electron microscopic immunolabeling with gold colloidal particles. The protein molecules were found to be localized uniformly in oocyte yolk platelets, but not in other cytoplasmic organelles. These findings suggest a novel role of yolk platelets as a reservoir for inductive signals transported by vitellogenin in the differentiation and patterning of cells in Xenopus embryos. (C) 1994 Academic Press, Inc.

DOI： 10.1006/bbrc.1994.1954

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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 isoforms 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. © 1994 by Academic Press, Inc.

DOI： 10.1006/dbio.1994.1143

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Xenopus follistatin and activins were purified from a Xenopus laevis cell line (XTC-F1) by four purification steps consisting of consecutive affinity chromatography on dextran sulfate-Sepharose and Sulfate Cellulofine, fast protein liquid chromatography gel permeation, and reverse-phase high performance liquid chromatography (HPLC). Our results thus obtained indicated that almost equimolar amounts of activins A, AB, and B were found to be present as a complex with follistatin (activin-binding protein) in the conditioned medium of XTC-F1 cells. Reverse-phase HPLC of the complex gave Xenopus follistatin and activins A, AB, and B. The purified Xenopus follistatin showed four major bands in a molecular mass range from 34 to 39 kDa by SDS-polyacrylamide gel electrophoresis under nonreducing conditions. The ability of each form of the protein to specifically bind activin was determined by activin-binding assay and ligand blotting analysis. Each protein was found to have the same NH2-terminus and its sequence was very homologous to that of mammalian follistatin. Several criteria including immunoblotting analysis and various functional assays revealed the existence of three isoforms of activins A, AB, and B in Xenopus, as in mammals. Xenopus activins significantly induced both ventral and dorsal mesoderm in explants of Xenopus blastula cells that would otherwise form epidermis. In a dose-dependent manner of each isoform of activin, the induced explants were able to differentiate into blood-like cells, coelomic epithelium, mesenchyme, muscle, and notochord. The induction patterns of three Xenopus activins were essentially the same. The mesoderm induction by the purified Xenopus activins was shown to be inhibited stoichiometrically by the purified Xenopus follistatin. These results indicate that Xenopus XTC-F1 cells secrete several molecular forms of follistatin/activin-binding protein and three isoforms of activins AB and B in addition to activin A. © 1993 by Academic Press, Inc.

DOI： 10.1006/dbio.1993.1227

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Language：English   Publisher：BLACKWELL SCIENCE  

DOI： 10.1111/j.1440-169X.1993.00123.x

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Language：Japanese   Publisher：共立出版  

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Publishing type：Book review, literature introduction, etc.  

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

To examine whether activin binds to follistatin, an activin-binding protein, to form a complex in vivo, we attempted to purify activin-follistatin complex from porcine follicular fluid. Our results thus obtained indicated that almost equimolar amounts of activins A, AB, and B are present as a complex with follistatin in the follicular fluid. Reverse-phase high performance liquid chromatography of the purified complex yielded follistatin and activins A, AB, and B. The activity of the purified activin B was found to be significantly lower than those of other activins in various assay systems such as stimulation of follicle-stimulating hormone secretion, induction of erythrodifferentiation, and potentiation of expression of gonadotropin receptors on ovarian cells. Moreover, binding of I-125-activin A to erythroleukemic cells which are activin-responsive was competed by activin B with approximately 10-fold lower potency compared with other activins. In contrast to these results, activin B was proved to have a potent Xenopus mesoderm-inducing activity, comparable with that of other activins. This indicates that, unlike activins A and AB, activin B can only elicit mesoderm-inducing activity and cannot function in other biological systems, suggesting a specific role of activin B in early development and unknown biological functions.

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Language：English   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

DOI： 10.1016/0006-291X(92)91199-Z

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Language：English   Publisher：Zoological Society of Japan  

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Other Link： http://id.nii.ac.jp/1141/00031648/

Publishing type：Book review, literature introduction, etc.  

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Language：English   Publisher：NATL ACAD SCIENCES  

Activin A, a member of the transforming growth factor-beta superfamily, has recently been found to have potent mesoderm-inducing activity on isolated early Xenopus animal-cap cells. We measured the activin activity of the Xenopus egg extract by using an erythroid-differentiating test with Friend leukemia cells. The results showed that an activin homologue is, indeed, contained in unfertilized eggs and blastulae of Xenopus laevis in a considerable amount. This activity was eluted at the same retention time as human activin A when fractionated by reversed-phase HPLC. Furthermore, the fraction containing erythroid-differentiating factor activity had mesoderm-inducing activity on Xenopus animal-cap cells. The mesoderm-inducing activity of this fraction was suppressed when coincubated with follistatin, an activin-binding protein. These results suggest that an endogenous activin may be a natural mesoderm-inducing factor acting in Xenopus embryogenesis.

DOI： 10.1073/pnas.88.15.6511

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Language：English   Publisher：ELSEVIER SCIENCE BV  

The erythroid differentiation capacity of the HPLC-purified mesoderm- and endoderm-inducing vegetalizing factor from chicken embryos and of recombinant erythroid differentiation factor (EDF = activin A), an evolutionary highly conserved member of the TGF-beta protein superfamily have been compared. Both factors stimulate the synthesis of hemoglobin in erythroleukemia cells in the same concentration range. The EDF-activity of the mesoderm-inducing HPLC-fractions is inhibited by follistatin, an EDF-binding protein. The factor induces in ectoderm of Triturus taeniatus all kinds of mesodermal organs. The wide spectrum of organs is very likely to be induced by secondary interactions. At higher concentration (15 ng/ml), notochord- and endoderm-like tissues are induced in a high percentage.

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Language：Japanese   Publisher：日本生化学会  

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We examined the quality of mesoderm induced by the action of activin A on the Xenopus presumptive ectoderm when various concentrations and treatment times were employed. The minimum concentration of activin A to induce mesodermal tissues was inversely proportional to its treatment time. The explants differentiated into different types of mesodermal tissues, from ventral-type to dorsal-type depending on the concentration of activin A and its treatment time. To confirm whether activin A has a role in establishing axial organization, activin A was injected into the blastocoel of late blastulae. About 70% of the injected embryos formed secondary tail-shaped outgrowths in which muscle and neural tube differentiated. The amount of activin A to form secondary outgrowths was 0.5-2.5 pg, roughly consistent with the amount estimated from in vitro experiments. As we have detected almost the same amount of activin homologue in the early embryos (Asashima et al., 1991 a), we speculate that activin A may be the natural mesodermal inducer, and that it is responsible for establishing axial organization in the Xenopus embryo.

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Language：English   Publisher：SPRINGER VERLAG  

The induction of mesoderm is an important process in early amphibian development. In recent studies, activin has become an effective candidate for a natural mesoderm-inducing factor. In the present study, we show that follistatin, an activin-binding protein purified from porcine ovary, inhibits the mesoderm-inducing activity of recombinant human activin A (rh activin A), which is identical to the erythroid differentiation factor (EDF). The quantity of follistatin required for effective suppression of activin was more than three-fold that of activin (w:w). Follistatin also inhibited the mesoderm-inducing activity of the vegetalizing factor purified from chick embryos, suggesting that the vegetalizing factor is closely related to activin.

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Language：English   Publisher：SPRINGER VERLAG  

Human recombinant activin A, which is identical with erythroid differentiation factor (EDF), was tested for its mesoderm-inducing activity in concentrations from 0.3-50 ng/ml, using ectoderm of Xenopus late blastula (Stage 9) as the responding tissue. At a low concentration of activin A, blood-like cells, mesenchyme, and coelomic epithelium were induced; at a moderate concentration muscle and neural tissue, and at a high concentration notochord. Activin A thus induced all mesodermal tissues in a dose-dependent manner, such that a low dose induced ventral structures and a high dose induced dorsal structures. Activin may act as an intrinsic inducing molecule responsible for establishing the dorso-ventral axis in early Xenopus development.

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Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：SPRINGER VERLAG  

DOI： 10.1007/BF01135742

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Language：English   Publisher：Zoological Society of Japan  

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