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ABSTRACT: 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.
Developmental Biology 10/2010; 346(2):170-80. · 4.07 Impact Factor
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ABSTRACT: 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.
Biochemical and Biophysical Research Communications 10/2010; 400(4):461-5. · 2.48 Impact Factor
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Yuko Aihara,
Yohei Hayashi,
Mitsuhi Hirata,
Nobutaka Ariki,
Shinsuke Shibata,
Narihito Nagoshi,
Mio Nakanishi,
Kiyoshi Ohnuma,
Masaki Warashina,
Tatsuo Michiue, Hideho Uchiyama,
Hideyuki Okano,
Makoto Asashima,
Miho Kusuda Furue
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ABSTRACT: 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 stem (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.
The International journal of developmental biology 01/2010; 54(8-9):1287-94. · 2.16 Impact Factor
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Sarkar M.A. Kawsar,
Ryo Matsumoto,
Yuki Fujii,
Hidetaro Yasumitsu, Hideho Uchiyama,
Masahiro Hosono,
Kazuo Nitta,
Jiharu Hamako,
Taei Matsui,
Noriaki Kojima,
Yasuhiro Ozeki
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ABSTRACT: The glycan-binding profile of a β-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 (GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-4Galβ1-4Glc) was found to exhibit the strongest ligand binding to the galectin while Forssman antigen (GalNAcα1-3GalNAcβ1-3Galα1- 4Galβ1-4Glc) and type-A-tetrasaccharide (GalNAcα1-3(Fucα1-2)Galβ1-4GlcNAcβ1-4Glc) 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.
Protein and Peptide Letters 05/2009; 16(6):677-684. · 1.94 Impact Factor
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Sarkar M A Kawsar,
Ryo Matsumoto,
Yuki Fujii,
Hidetaro Yasumitsu, Hideho Uchiyama,
Masahiro Hosono,
Kazuo Nitta,
Jiharu Hamako,
Taei Matsui,
Noriaki Kojima,
Yasuhiro Ozeki
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ABSTRACT: 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 (GalNAcalpha1-3(Fucalpha1-2)Galbeta;1-4GlcNAcbeta1-4Galbeta1-4Glc) was found to exhibit the strongest ligand binding to the galectin while Forssman antigen (GalNAcalpha1-3GalNAcbeta1-3Galalpha1-4Galbeta1-4Glc) and type-A-tetrasaccharide (GalNAcalpha1-3(Fucalpha1-2)Galbeta1-4GlcNAcbeta1-4Glc) 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.
Protein and Peptide Letters 02/2009; 16(6):677-84. · 1.94 Impact Factor
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ABSTRACT: 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.
Developmental Dynamics 01/2009; 237(12):3749-61. · 2.54 Impact Factor
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ABSTRACT: 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/Ripply family 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.
The International journal of developmental biology 01/2009; 53(4):631-9. · 2.16 Impact Factor
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ABSTRACT: 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, mespbDeltaDBD, 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.
Biochemical and Biophysical Research Communications 09/2008; 372(4):607-12. · 2.48 Impact Factor
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ABSTRACT: Correlation between activin and retinoic acid (RA), both of which affect early amphibian development, was studied using Xenopus laevis embryos. In the first set of experiments, two isomers of RA, all-trans RA and 13-cis RA, were compared in terms of stability of biological activity against light. Xenopus blastulae were dipped in RA solutions which had either been kept away from light, or had been exposed to light for a few hours. At doses ranging from 10–4to 10–6M, RA elicited head deformity. All-trans RA, under both dark and light conditions, had similarly potent effects. On the other hand, 13-cis RA under dark conditions had much weaker effects than it did under light conditions.In the second set of experiments, activin was mixed with all-trans RA, and the inducing effects on the animal cap explants were investigated. Activin at a concentration of 10 ng/ml induced notochord. In combination with 10–6M RA, muscle was well induced instead of notochord. In combination with 10–5M RA, pronephric tubules were markedly induced. Pronephric tubules were never induced by activin alone, at any of the various concentrations employed. This is the first report on the very high frequency of induction of pronephric tubules by the combination of activin A and all-trans RA in the Xenopus ectoderm.
Embryologia 07/2008; 35(2):123 - 128. · 2.21 Impact Factor
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ABSTRACT: 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.
Developmental Biology 02/2008; 313(2):816-28. · 4.07 Impact Factor
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ABSTRACT: 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.
The International Journal of Developmental Biology 02/2006; 50(8):681-9. · 2.82 Impact Factor
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ABSTRACT: 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.
Embryologia 01/2006; 47(9):581-9. · 2.21 Impact Factor
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ABSTRACT: 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.
Developmental Biology 02/2004; 265(2):355-68. · 4.07 Impact Factor
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ABSTRACT: 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.
Archiv für Entwickelungsmechanik der Organismen 08/1991; 200(4):230-233. · 1.77 Impact Factor
