Appropriate Amounts and Activity of the Wilms Tumor Suppressor Gene, wt1, Are Required for Normal Pronephros Development of Xenopus Embryos


Application of the RNA interference technique to Xenopus embryos: Specific reduction of the β-catenin gene products by short double-stranded RNA produced by recombinant human Dicer

RNA interference (RNAi) is a technique for suppressing the function of specific genes and is widely used in many organisms, including yeast, nematodes, flies, plants, mice, and cultured mammalian cells. As of date, this technique has not been successfully applied to Xenopus laevis embryos. In this study, we applied RNAi to Xenopus embryos using β-catenin as a model gene. Injection of long double-stranded RNA (dsRNA) corresponding to the 3'-untranslated region of β-catenin mRNA into embryos induced embryonic lethality without any specific phenotype. However, injection of short dsRNA, generated from long dsRNA by treatment with recombinant human Dicer, into embryos resulted in decreased expression of endogenous β-catenin mRNA and protein, as well as decreased Wnt signaling activity in the embryos. The decrease in β-catenin mRNA and protein levels was observed only after mid-blastula transition. Embryos injected with short dsRNA showed a characteristic phenotype of enlarged anterior structures and loss of posterior structures. These phenotypes, as well as the increased expression of the anterior gene and decreased expression of the posterior gene, suggest that RNAi against the β-catenin gene suppresses the "late Wnt signaling" involved in proper anterior-posterior patterning of Xenopus embryos. The effect of RNAi on Xenopus embryos was also found to be sensitive to temperature. These results strongly suggest that the RNAi technique can be applied to Xenopus embryos using short dsRNAs, appropriate temperature control, and proper selection of target genes.


Involvement of Myt1 kinase in the G2 phase of thefirst cell cycle inXenopus laevis

During cleavage of Xenopus laevis, the first mitotic cell cycle immediately following fertilization is approximately 90 min and consists of S, G2, and M phases. In contrast, the subsequent eleven cell cycles are approximately 30 min and consist mostly of S and M phases. The balance between Cdc25 and Wee1A/Myt1 is thought to be crucial for Xenopus first cell cycle progression; however, the role of Myt1 in this period has not been fully investigated. In this study, we examined the roles of Myt1, Wee1A, and Cdc25A in the first cell cycle of Xenopus laevis. Inhibition of Cdc25A with antisense morpholino oligonucleotides lengthened the duration of the first cell cycle to some extent, whereas it was slightly shortened by ectopic Cdc25A expression, suggesting that the low concentration of Cdc25A during the first cell cycle does not fully account for the long duration of this cycle. Using the Wee1A antisense morpholino oligonucleotide and neutralizing antibody against Myt1, we found that Myt1 phosphorylates and inhibits Cdk1 much more effectively than Wee1A during the first cell cycle in Xenopus. Taken together, these results suggest that the activity of Myt1 is predominantly responsible for the duration of the long G2 phase in the first mitotic cell cycle in Xenopus.

Biochemical and Biophysical Research Communications


Conservatism and variability of gene expression profiles among homeologous transcription factors in Xenopus laevis.

Xenopus laevis has an allotetraploid genome of 3.1Gb, 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 otx1 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.


Genome evolution in the allotetraploid frog Xenopus laevis.

To explore the origins and consequences of tetraploidy in the African clawed frog, we sequenced the Xenopus laevis genome and compared it to the related diploid X. tropicalis genome. We characterize the allotetraploid origin of X. laevis by partitioning its genome into two homoeologous subgenomes, marked by distinct families of 'fossil' transposable elements. On the basis of the activity of these elements and the age of hundreds of unitary pseudogenes, we estimate that the two diploid progenitor species diverged around 34 million years ago (Ma) and combined to form an allotetraploid around 17-18 Ma. More than 56% of all genes were retained in two homoeologous copies. Protein function, gene expression, and the amount of conserved flanking sequence all correlate with retention rates. The subgenomes have evolved asymmetrically, with one chromosome set more often preserving the ancestral state and the other experiencing more gene loss, deletion, rearrangement, and reduced gene expression.


A potential molecular pathogenesis of cardiac/laterality defects in Oculo-Facio-Cardio-Dental syndrome.

Pitx2 is the last effector of the left-right (LR) cascade known to date and plays a crucial role in the patterning of LR asymmetry. In Xenopus embryos, the expression of Pitx2 gene in the left lateral plate mesoderm (LPM) is directly regulated by Xnr1 signaling, which is mediated by Smads and FoxH1. Previous studies suggest that the suppression of Pitx2 gene in the left LPM is a potential cause of cardiac/laterality defects in Oculo-Facio-Cardio-Dental (OFCD) syndrome, which is known to be caused by mutations in BCL6 co-repressor (BCOR) gene. Recently, our work has revealed that the BCL6/BCOR complex blocks Notch-dependent transcriptional activity to protect the expression of Pitx2 in the left LPM from the inhibitory activity of Notch signaling. These studies indicated that uncontrolled Notch activity in the left LPM caused by dysfunction of BCOR may result in cardiac/laterality defects of OFCD syndrome. However, this Notch-dependent inhibitory mechanism of Pitx2 gene transcription still remains unknown. Here we report that transcriptional repressor ESR1, which acts downstream of Notch signaling, inhibits the expression of Pitx2 gene by binding to a left side-specific enhancer (ASE) region in Pitx2 gene and recruiting histone deacetylase 1 (HDAC1) to this region. Once HDAC1 is tethered, histone acetyltransferase p300 is no longer recruited to the Xnr1-dependent transcriptional complex on the ASE region, leading to the suppression of Pitx2 gene in the left LPM. The study presented here uncovers the regulatory mechanism of Pitx2 gene transcription which may contribute to an understanding of pathogenesis of OFCD syndrome.


Hypergravity Specifically Affects Anterior Head Formation in Early Xenopus Embryos


Analysis of Head-Defects Caused by Hypergravity in Early Xenopus Embryos


Hypergravity specifically effects head formation in the Xenopus embryos raised under hypergravity condition

過重力はアフリカツメガエルの頭部形成(特に眼と脳)に特異的に影響を与える


過重力によって生じたアフリカツメガエル初期胚の頭部形成異常の解析 II

過重力によって生じたアフリカツメガエル初期胚の頭部形成異常の解析 II


A Report from Experience-based Study Programs of Life Science for High School Students by University-High School Cooperation: Examples of SPP and SSH Programs. --- ∼SPPおよびSSHを例として∼

高大連携による高校生への生命科学体験型講座の実施報告 --- ∼SPPおよびSSHを例として∼

Journal of University Education Research


Effect of strong static magnetic fields on amphibian life cycle-morphological and milecular biological analysis of early development-

両生類の生活環における強磁場の影響ー初期発生の形態学的および分子生物学的解析ー


過重力下で見られるアフリカツメガエル頭部形成異常の解析

過重力下で見られるアフリカツメガエル頭部形成異常の解析


Novel S-S loops in the globin chains from the giant hemoglobin of the polychaete Perinereis aibuhitensis.


FoxM1-driven cell division is required for neuronal differentiation in early Xenopus embryos.

In vertebrate embryogenesis, neural induction is the earliest step through which the fate of embryonic ectoderm to neuroectoderm becomes determined. Cells in the neuroectoderm or neural precursors actively proliferate before they exit from the cell cycle and differentiate into neural cells. However, little is known about the relationship between cell division and neural differentiation, although, in Xenopus, cell division after the onset of gastrulation has been suggested to be nonessential for neural differentiation. Here, we show that the Forkhead transcription factor FoxM1 is required for both proliferation and differentiation of neuronal precursors in early Xenopus embryos. FoxM1 is expressed in the neuroectoderm and is required for cell proliferation in this region. Specifically, inhibition of BMP signaling, an important step for neural induction, induces the expression of FoxM1 and its target G2-M cell-cycle regulators, such as Cdc25B and cyclin B3, thereby promoting cell division in the neuroectoderm. Furthermore, G2-M cell-cycle progression or cell division mediated by FoxM1 or its target G2-M regulators is essential for neuronal differentiation but not for specification of the neuroectoderm. These results suggest that FoxM1 functions to link cell division and neuronal differentiation in early Xenopus embryos.


過重力によるニシツメガエル脳下垂体‐甲状腺系の絹小低下に対する甲状腺ホルモン投与の効果


アフリカツメガエル卵成熟過程に対する過重力の影響II

アフリカツメガエル卵成熟過程に対する過重力の影響II


Effects of Hypergravity on Pituitary-Target Organs in the Frog, Xenopus laevis


Effects on hypergravity on pituitary-target organ axes in the frog, Xenopus laevis

過重力環境がアフリカツメガエルの脳下垂体‐標的器官系におよぼす影響についての研究


Effects of the hypergravity on oocyte maturation of Xenopus laevis

アフリカツメガエル卵成熟過程に対する過重力の影響


Effects of gravity on the amphibian life cycle - morphological and molecular biological analyses of early development, morphogenesis and metamorphosis

両生類の生活環に対する重力影響ー初期発生，形態形成や変態の形態学的および分子生物学的解析


Regulation of the Lim-1 gene is mediated through conserved FAST-1/FoxH1 sites in the first intron

The Lim-1 gene encodes a LIM-homeodomain transcription factor that is highly conserved among vertebrates and is required for successful gastrulation and head formation. The expression of this gene in the mesoderm of the gastrula is known to require an activin/nodal signal. Earlier studies have shown that the Xenopus Lim-1 (Xlim-1) gene contains an activin response element (ARE) in its first intron, which cooperates with an activin-unresponsive upstream promoter in the regulation of the gene. Here, we show that the Xlim-1 ARE contains a cluster of FAST-1/FoxH1 and Smad4 recognition sites; such sites have been shown to mediate activin/nodal responses in other genes. By using reporter constructs with mutated FAST-1/FoxH1 sites and FAST-1/FoxH1 protein chimeras, we show that the regulation of Xlim-1 by activin depends on FAST-1/FoxH1 function. Comparative studies on the zebrafish lim1 gene indicate the presence of FoxH1 sites in the first intron of this gene and provide evidence for the requirement for FoxH1 function in its regulation. These results illuminate the conserved nature of the transcriptional regulation of the Lim-1 gene in different vertebrate animals.


Two step regulation of left-right asymmetric Pitx2 expression: Initiation by Nodal signaling and maintenance by Nkx2

Pitx2 is left--right (L--R) asymmetrically expressed initially in the lateral plate and later in primordial visceral organs. The transcriptional regulatory mechanisms that underlie L--R asymmetric expression of Pitx2 were investigated. Mouse Pitx2 has a left side-specific enhancer (ASE) that mediates both the initiation and maintenance of L--R asymmetric expression. This element contains three binding sites for the transcription factor FAST. The FAST binding sites function as Nodal-responsive elements and are sufficient for the initiation but not for the maintenance of asymmetric expression. The maintenance requires an Nkx2-5 binding site also present within the ASE. These results suggest that the left-sided expression of Pitx2 is directly initiated by Nodal signaling and is subsequently maintained by Nkx2. Such two-step control may represent a general mechanism for gene regulation during development.


Activin/Nodal responsiveness and asymmetric expression of a Xenopus nodal-related gene converge on a FAST-regulated module in intron 1

Vertebrate Nodal-related factors play central roles in mesendoderm induction and left-right axis specification, but the mechanisms regulating their expression are largely unknown. We identify an element in Xnr1 intron 1 that is activated by activin and Vg1, autoactivated by Xnrs, and suppressed by ventral inducers like BMP4. Intron 1 contains three FAST binding sites on which FAST/Smad transcriptional complexes can assemble; these sites are differentially involved in intron 1-mediated reporter gene expression. Interference with FAST function abolishes intron 1 activity, and transcriptional activation of Xnrs by activin in embryonic tissue explant assays, identifying FAST as an essential mediator of Xnr autoregulation and/or 'signal relay' from activin-like molecules. Furthermore, the mapping of endogenous activators of the Xnr1 intronic enhancer within Xenopus embryos agrees well with the pattern of Xnr1 transcription during embryogenesis. In transgenic mice, Xnr1 intron 1 mimics a similarly located enhancer in the mouse nodal gene, and directs FAST site-dependent expression in the primitive streak during gastrulation, and unilateral expression during early somitogenesis. The FAST cassette is similar in an ascidian nodal-related gene, suggesting an ancient origin for this regulatory module. Thus, an evolutionarily conserved intronic enhancer in Xnr1 is involved in both mesendoderm induction and asymmetric expression during left-right axis formation.


Left-right asymmetric expression of lefty2 and nodal is induced by a signaling pathway that includes the transcription factor FAST2

The left-right (L-R) asymmetric expression of lefty2 and nodal is controlled by a left side-specific enhancer (ASE). The transcription factor FAST2, which can mediate signaling by TGF beta and activin, has now been identified as a protein that binds to a conserved sequence in ASE. These FAST2 binding sites were both essential and sufficient for L-R asymmetric gene expression. The Fast2 gene is bilaterally expressed when nodal and lefty2 are expressed on the left side. TGF beta and activin can activate the ASE activity in a FAST2-dependent manner, while Nodal can do so in the presence of an EGF-CFC protein. These results suggest that the asymmetric expression of lefty2 and nodal is induced by a left side-specific TGF beta-related factor, which is most likely Nodal itself.


FAST-1 is a key maternal effector of mesodermal inducers in the early Xenopus embryo

We have examined the role of the maternally encoded transcription factor FAST-1 in the establishment of the mesodermal transcriptional program in Xenopus embryos. FAST-1 has been shown to associate with Smad2 and Smad4, transducers of TGFbeta superfamily signals, in response to stimulation by several TGFbeta superfamily ligands. The FAST-1/Smad2/Smad4 complex binds and activates a 50 bp activin responsive element identified in the promoter of the meso-endodermal marker Mix.2. We have now used three complementary approaches to demonstrate that FAST-1 is a central regulator of mesoderm induction by ectopic TGFbeta superfamily ligands and during endogenous patterning: ectopic expression of mutationally activated FAST-1, ectopic expression of dominant inhibitory FAST-1, and injection of a blocking antibody specific for FAST-1. Expression of constitutively transcriptionally active FAST-1 fusion protein (FAST-VP16(A)) in prospective ectoderm can directly induce the same set of general and dorsal mesodermal genes, as well as some endodermal genes, as are induced by activin or Vg1. In intact embryos, this construct can induce secondary axes similar to those induced by activin or Vg1. Conversely, expression of a FAST-1-repressor fusion (FAST-En(R)) in prospective ectoderm blocks induction of mesodermal genes by activin, while expression of FAST-En(R) in intact embryos prevents general/dorsal mesodermal gene expression and axial development. Injection of a blocking antibody specific for FAST-1 prevents induction of mesodermal response genes by activin or Vg1, but not by FGF. In intact embryos, this antibody can prevent the expression of early mesodermal markers and inhibit axis formation, demonstrating that FAST-1 is a necessary component of the first steps in the specification of mesoderm.


The role of transcription factors involved in TGFß superfamily signaling during development

Recent studies of transforming growth factor-beta (TGF-beta) signaling have identified a signaling pathway that includes Ser/Thr kinase transmembrane receptors, intracellular substrates and transducers of receptor activation known as Smads, and DNA-binding transcription factors that are regulated by interaction with Smads. Both genetic and biochemical studies show that Smads are central mediators of TGF-beta signaling. How do Smads regulate the expression of target genes in the nucleus? Over the past three years, transcription factors involved in TGF-beta signaling have been identified and the molecular events in the nucleus have begun to be understood. Both Smads, which have intrinsic DNA binding activity, and additional transcription factors, act together to regulate the expression of target genes in the nucleus. Smads are relatively ubiquitously expressed in embryos during the development, while interacting transcription factors are expressed with a restricted pattern, either temporally or spatially. Therefore the developmental specificity of TGF-beta signaling may be, at least in part, determined by cell-type specific transcription factors.


Drosophila CtBP: a Hairy-interacting protein required for embryonic segmentation and Hairy-mediated transcriptional repression


Smad4 and FAST-1 in the assembly of activin-responsive factor

Members of the TGF-beta superfamily of signalling molecules work by activating transmembrane receptors with phosphorylating activity (serine-threonine kinase receptors); these in turn phosphorylate and activate SMADs, a class of signal transducers. Activins are growth factors that act primarily through Smad2, possibly in partnership with Smad4, which forms heteromeric complexes with different ligand-specific SMADs after activation. In frog embryos, Smad2 participates in an activin-responsive factor (ARF), which then binds to a promoter element of the Mix.2 gene. The principal DNA-binding component of ARF is FAST-1, a transcription factor with a novel winged-helix structure. We now report that Smad4 is present in ARF, and that FAST-1, Smad4 and Smad2 co-immunoprecipitate in a ligand-regulated fashion. We have mapped the site of interaction between FAST-1 and Smad2/Smad4 to a novel carboxy-terminal domain of FAST-1, and find that overexpression of this domain specifically inhibits activin signalling. In a yeast two-hybrid assay, the FAST-1 carboxy terminus interacts with Smad2 but not Smad4. Deletion mutants of the FAST-1 carboxy terminus that still participate in ligand-regulated Smad2 binding no longer associated with Smad4 or ARF. These results indicate that Smad4 stabilizes a ligand-stimulated Smad2-FAST-1 complex as an active DNA-binding factor.


General interaction of DED1 encoding a putative ATP-dependent RNA helicase with SRM1 encoding a mammalian RCC1 homologue in Saccharomyces cerevisiae


Functional equivalence of human X- and Y-encoded isoforms of ribosomal protein S4 consistent with a role in Turner syndrome

Several genes are found on both the human X and Y chromosomes in regions that do not recombine during male meiosis. In each case, nucleotide sequence analysis suggests that these X-Y gene pairs encode similar but nonidentical proteins. Here we show that the human Y- and X-encoded ribosomal proteins, RPS4Y and RPS4X, are interchangeable and provide an essential function: either protein rescued a mutant hamster cell line that was otherwise incapable of growth at modestly elevated temperatures. These findings are consistent with the hypothesis that RPS4 deficiency has a role in Turner syndrome, a complex human phenotype associated with monosomy X.


Nucleotide sequence of Xenopus homeobox gene, En-1


Molecular cloning of the human gene, CCG2, that complements the BHK-derived temperature-sensitive cell cycle mutant tsBN63: identification of CCG2 with the human X chromosomal SCAR/RPS4 gene


Characteristics of hybrids between Taraxacum japonicum and T. officinale (Asteraceae, Asterales) growing in urban parks of Tokushima city, western Japan

徳島市内の都市的緑地に生育するカンサイタンポポとセイヨウタンポポの 雑種について


University and High school cooperation project-A report from experience-based study program of high school students in the laboratory of the university-

高大連携事業「高校生の大学研究室への体験入学型学習プログラム」の実施報告

Journal of University Education Research


A practical training in enveronmental education for traversal thinking of a nature and historical heritage of headstream of Yoshino river;a educational significance of environmental education for symbiosis --- 現代GP「豊穣な吉野川を持続可能とする共生環境教育」の一環として

現代GP体験ゼミ 吉野川源流の自然と文化を見て・知って・考えてみる --- 現代GP「豊穣な吉野川を持続可能とする共生環境教育」の一環として


Comparative study on the primary structure of globin chains of the extracellular giant hemoglobin from the polychaet Perinereis aibuhitensis and Tylonrrhynchus heterochaetus

Natural Science Research, Faculty of Integrated Arts and Sciences, The University of Tokushima

徳島大学総合科学部自然科学研究


高校生向け課題研究研修会によるアクティブ・ラーニング型高大連携とFDへの展開

高校生向け課題研究研修会によるアクティブ・ラーニング型高大連携とFDへの展開

Education Conference, the University of Tokushima, Abstracts(2016)

平成28年度全学FD 徳島大学教育カンファレンス in 徳島 発表抄録集


アクティブ・ラーニングを取り入れた授業実践

アクティブ・ラーニングを取り入れた授業実践

平成28年度徳島大学「大学教育再生加速プログラム」シンポジウム アクティブ・ラーニング&反転授業 発表抄録集


A report from experience-based study program of high school students in the laboratory of the university (4)

高大連携事業「高校生の大学研究室への体験入学型学習プログラム」実施報告(第4報)

Education Conference, the University of Tokushima, Abstracts(2012)

平成24年度全学FD 徳島大学教育カンファレンス in 徳島 発表抄録集


A report from experience-based study program of high school students in the laboratory of the university (3)

高大連携事業「高校生の大学研究室への体験入学型学習プログラム」実施報告(第3報)

Education Conference, the University of Tokushima, Abstracts(2011)

平成23年度全学FD 徳島大学教育カンファレンス in 徳島 発表抄録集


高大連携事業「高校生の大学研究室への体験入学型学習プログラム」実施報告

高大連携事業「高校生の大学研究室への体験入学型学習プログラム」実施報告

平成20年度全学FD徳島大学教育カンファレンス 発表抄録集

平成20年度全学FD徳島大学教育カンファレンス 発表抄録集