担当区分：最終著者,　責任著者   記述言語：その他  

DOI： 10.1101/2023.11.27.568629

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記述言語：その他  

Cell states are regulated by the response of signaling pathways to receptor ligand-binding and inter-cellular interactions. High-resolution imaging has been attempted to explore the dynamics of these processes and, recently, multiplexed imaging has profiled cell states by achieving comprehensive acquisition of spatial protein information from cells. However, resolution is still compromised when visualizing activated signals. Here we developed Precise Emission Canceling Antibody (PECAb) that have cleavable fluorescent labeling. PECAbs allow high-resolution sequential imaging using hundreds of antibodies and allow reconstruction of the spatiotemporal dynamics of signaling pathways. Additionally, combining this approach with seq-smFISH can effectively classify cells and identify their signal activation states in human tissue. Overall, the PECAb system can serve as a comprehensive platform for analyzing complex cell processes.

DOI： 10.1101/2023.10.17.561810

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担当区分：筆頭著者,　責任著者   記述言語：英語   出版者・発行元：Current Opinion in Structural Biology  

Higher-order genomic structures play a critical role in regulating gene expression by influencing the spatial proximity of promoters and enhancers. Live-cell imaging studies have demonstrated that three-dimensional genome structures undergo dynamic changes over time. Transcription is also dynamic, with genes frequently switching between active and inactive states. Recent observations suggest that the formation of condensates, composed of transcription-related factors, RNA, and RNA-binding proteins, around genes can regulate transcription. Advancements in technology have facilitated the visualization of the intricate spatiotemporal relationship between higher-order genomic structures, condensate formation, and transcriptional activity in living cells.

DOI： 10.1016/j.sbi.2023.102615

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担当区分：最終著者,　責任著者   記述言語：その他   掲載種別：研究論文（学術雑誌）   出版者・発行元：Nature Communications  

転写は動的な過程である。RNAポリメラーゼIIとコアクチベーターのタンパク質クラスター、遺伝子座、転写活性の動的な関係を検出するために、転写開始部位のすぐ下流にMS2リピート、TetOリピート、選択マーカー付きインテインを挿入する。個々のエレメントを最適化することで、私たちはSpliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag)システムを開発した。マウス胚性幹細胞でNanog遺伝子が転写される際、RNAポリメラーゼIIとBRD4のクラスターが転写開始点近傍に観察される。対照的に、MED19とMED22のクラスターは、転写活性がなくても転写開始部位の近くに位置する傾向がある。このように、STREAMING-tagシステムは、転写活性と遺伝子近傍のタンパク質クラスターとの時空間的関係を明らかにする。この強力なツールは、生きた細胞における転写制御を定量的に理解するのに有用である。

DOI： 10.1038/s41467-022-35286-2

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

The nucleolus is the site of ribosome assembly and formed through liquid-liquid phase separation. Multiple ribosomal DNA (rDNA) arrays are bundled in the nucleolus, but the underlying mechanism and significance are unknown. In the present study, we performed high-content screening followed by image profiling with the wndchrm machine learning algorithm. We revealed that cells lacking a specific 60S ribosomal protein set exhibited common nucleolar disintegration. The depletion of RPL5 (also known as uL18), the liquid-liquid phase separation facilitator, was most effective, and resulted in an enlarged and un-separated sub-nucleolar compartment. Single-molecule tracking analysis revealed less-constrained mobility of its components. rDNA arrays were also unbundled. These results were recapitulated by a coarse-grained molecular dynamics model. Transcription and processing of ribosomal RNA were repressed in these aberrant nucleoli. Consistently, the nucleoli were disordered in peripheral blood cells from a Diamond-Blackfan anemia patient harboring a heterozygous, large deletion in RPL5. Our combinatorial analyses newly define the role of RPL5 in rDNA array bundling and the biophysical properties of the nucleolus, which may contribute to the etiology of ribosomopathy.

DOI： 10.26508/lsa.202101045

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

Trisomy 21, 18, and 13 are the major autosomal aneuploidy disorders in humans. They are mostly derived from chromosome non-disjunction in maternal meiosis, and the extra trisomic chromosome can cause several congenital malformations. Various genes on the trisomic chromosomes are intricately involved in the development of disease, and fundamental treatments have not yet been established. However, chromosome therapy has been developed to correct the extra chromosome in cultured patient cells, and it was recently reported that during reprogramming into iPSCs, fibroblasts from a Down syndrome patient lost the extra chromosome 21 due to a phenomenon called trisomy-biased chromosome loss. To gain preliminary insights into the underlying mechanism of trisomy rescue during the early stages of reprogramming, we reprogrammed skin fibroblasts from patients with trisomy syndromes 21, 18, 13, and 9 to iPSC, and evaluated the genomes of the individual iPSC colonies by molecular cytogenetic techniques. We report the spontaneous correction from trisomy to disomy upon cell reprogramming in at least one cell line examined from each of the trisomy syndromes, and three possible combinations of chromosomes were selected in the isogenic trisomy-rescued iPSC clones. Single nucleotide polymorphism analysis showed that the trisomy-rescued clones exhibited either heterodisomy or segmental uniparental isodisomy, ruling out the possibility that two trisomic chromosomes were lost simultaneously and the remaining one was duplicated, suggesting instead that one trisomic chromosome was lost to generate disomic cells. These results demonstrated that trisomy rescue may be a phenomenon with random loss of the extra chromosome and subsequent selection for disomic iPSCs, which is analogous to the karyotype correction in early preimplantation embryos. Our finding is relevant for elucidating the mechanisms of autonomous karyotype correction and future application in basic and clinical research on aneuploidy disorders.

DOI： 10.1371/journal.pone.0264965

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記述言語：その他   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41594-020-0493-6

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

Transcriptional bursting is the stochastic activation and inactivation of promoters, contributing to cell-to-cell heterogeneity in gene expression. However, the mechanism underlying the regulation of transcriptional bursting kinetics (burst size and frequency) in mammalian cells remains elusive. In this study, we performed single-cell RNA sequencing to analyze the intrinsic noise and mRNA levels for elucidating the transcriptional bursting kinetics in mouse embryonic stem cells. Informatics analyses and functional assays revealed that transcriptional bursting kinetics was regulated by a combination of promoter- and gene body-binding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9-based screening identified that the Akt/MAPK signaling pathway regulated bursting kinetics by modulating transcription elongation efficiency. These results uncovered the key molecular mechanisms underlying transcriptional bursting and cell-to-cell gene expression noise in mammalian cells.

DOI： 10.1126/sciadv.aaz6699

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.15252/embj.2019103499

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1093/nargab/lqaa020

担当区分：最終著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1371/journal.pcbi.1007289

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.cell.2019.05.029

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The nuclear positioning and chromatin dynamics of eukaryotic genes are closely related to the regulation of gene expression, but they have not been well examined during early development, which is accompanied by rapid cell cycle progression and dynamic changes in nuclear organization, such as nuclear size and chromatin constitution. In this study, we focused on the early development of the sea urchin Hemicentrotus pulcherrimus and performed three-dimensional fluorescence in situ hybridization of gene loci encoding early histones (one of the types of histone in sea urchin). There are two non-allelic early histone gene loci per sea urchin genome. We found that during the morula stage, when the early histone gene expression levels are at their maximum, interchromosomal interactions were often formed between the early histone gene loci on separate chromosomes and that the gene loci were directed to locate to more interior positions. Furthermore, these interactions were associated with the active transcription of the early histone genes. Thus, such dynamic interchromosomal interactions may contribute to the efficient synthesis of early histone mRNA during the morula stage of sea urchin development.

DOI： 10.1242/jcs.206862

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Ligation-mediated polymerase chain reaction (LM-PCR) is a common technique for amplification of a pool of DNA fragments. Here, a double-stranded oligonucleotide consisting of two primer sequences in back-to-back orientation was designed as an adapter for LM-PCR. When DNA fragments were ligated with this adapter, the fragments were sandwiched between two adapters in random orientations. In the ensuing PCR, ligation products linked at each end to an opposite side of the adapter, i.e. to a distinct primer sequence, were preferentially amplified compared with products linked at each end to an identical primer sequence. The use of this adapter in LM-PCR reduced the impairment of PCR by substrate DNA with a high GC content, compared with the use of traditional LM-PCR adapters. This result suggested that our method has the potential to contribute to reduction of the amplification bias that is caused by an intrinsic property of the sequence context in substrate DNA. A DNA preparation obtained from a chromatin immunoprecipitation assay using pulldown of a specific form of histone H3 was successfully amplified using the modified LM-PCR, and the amplified products could be used as probes in a fluorescence in situ hybridization analysis. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ab.2017.05.011

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

The relationship between genome organization and gene expression has recently been established. However, the relationships between spatial organization, dynamics, and transcriptional regulation of the genome remain unknown. In this study, we developed a live-imaging method for simultaneous measurements of the transcriptional activity and nuclear position of endogenous genes, which we termed the 'Real-time Observation of Localization and EXpression (ROLEX)' system. We demonstrated that ROLEX is highly specific and does not affect the expression level of the target gene. ROLEX enabled detection of sub-genome-wide mobility changes that depended on the state of Nanog transactivation in embryonic stem cells. We believe that the ROLEX system will become a powerful tool for exploring the relationship between transcription and nuclear dynamics in living cells.

DOI： 10.1093/nar/gkv624

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The primary cilium is an antenna-like, microtubule-based organelle on the surface of most vertebrate cells for receiving extracellular information. Although primary cilia form in the quiescent phase, ciliary disassembly occurs when quiescent cells re-enter the proliferative phase. It was shown that a mitotic kinase, Polo-like kinase 1 (PLK1), is required for cell-proliferation-coupled primary cilia disassembly. Here, we report that kinesin superfamily protein 2A (KIF2A), phosphorylated at T554 by PLK1, exhibits microtubule-depolymerizing activity at the mother centriole to disassemble the primary cilium in a growth-signal-dependent manner. KIF2A-deficient hTERT-RPE1 cells showed the impairment of primary cilia disassembly following growth stimulation. It was also found that the PLK1-KIF2A pathway is constitutively active in cells from patients with premature chromatid separation (PCS) syndrome and is responsible for defective ciliogenesis in this syndrome. These findings provide insights into the roles of the PLK1-KIF2A pathway in physiological cilia disassembly and cilia-associated disorders.

DOI： 10.1016/j.celrep.2015.01.003

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

Mouse embryonic stem cells (mESCs) are self-renewing and capable of differentiating into any of the three germ layers. An interesting feature of mESCs is the presence of cell-to-cell heterogeneity in gene expression that may be responsible for cell fate decisions. Nanog, a key transcription factor for pluripotency, displays heterogeneous expression in mESCs, via mechanisms that are not fully understood. To understand this variability, we quantitatively analyzed Nanog transcription and found that Nanog was both infrequently transcribed, and transcribed in a pulsatile and stochastic manner. It is possible that such stochastic transcriptional activation could contribute to the heterogeneity observed in Nanog expression as "intrinsic noise.'' To discriminate the effects of both intrinsic noise from other (extrinsic) noise on the expression variability of Nanog mRNA, we performed allele-specific single-molecule RNA fluorescent in situ hybridization in a reporter cell line and found that intrinsic noise contributed to approximately 45% of the total variability in Nanog expression. Furthermore, we found that Nanog mRNA and protein levels were well correlated in individual cells. These results suggest that stochastic promoter activation significantly affects the Nanog expression variability in mESCs.

DOI： 10.1038/srep07125

リポジトリ公開URL： https://hdl.handle.net/2324/7237095

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

Cancer-prone syndrome of premature chromatid separation with mosaic variegated aneuploidy [PCS (MVA) syndrome] is a rare autosomal recessive disorder characterized by constitutional aneuploidy and a high risk of childhood cancer. We previously reported monoallelicmutations in the BUB1B gene (encoding BUBR1) in seven Japanese families with the syndrome. No second mutation was found in the opposite allele of any of the families studied, although a conserved BUB1B haplotype and a decreased transcript were identified. To clarify the molecular pathology of the second allele, we extended our mutational search to a candidate region surrounding BUB1B. A unique single nucleotide substitution, G > A at ss802470619, was identified in an intergenic region 44 kb upstream of a BUB1B transcription start site, which cosegregated with the disorder. To examine whether this is the causal mutation, we designed a transcription activator-like effector nuclease-mediated two-step single-base pair editing strategy and biallelically introduced this substitution into cultured human cells. The cell clones showed reduced BUB1B transcripts, increased PCS frequency, and MVA, which are the hallmarks of the syndrome. We also encountered a case of a Japanese infant with PCS (MVA) syndrome carrying a homozygous single nucleotide substitution at ss802470619. These results suggested that the nucleotide substitution identified was the causal mutation of PCS (MVA) syndrome.

DOI： 10.1073/pnas.1317008111

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Transcription activator-like effector (TALE) nuclease (TALEN) is a site-specific nuclease, which can be freely designed and easily constructed. Numerous methods of constructing TALENs harboring different TALE scaffolds and repeat variants have recently been reported. However, the functionalities of structurally different TALENs have not yet been compared. Here, we report on the functional differences among several types of TALENs targeting the same loci. Using HEK293T cell-based single-strand annealing and Cel-I nuclease assays, we found that TALENs with periodically-patterned repeat variants harboring non-repeat-variable di-residue (non-RVD) variations (Platinum TALENs) showed higher activities than TALENs without non-RVD variations. Furthermore, the efficiencies of gene disruption mediated by Platinum TALENs in frogs and rats were significantly higher than in previous reports. This study therefore demonstrated an efficient system for the construction of these highly active Platinum TALENs (Platinum Gate system), which could establish a new standard in TALEN engineering.

DOI： 10.1038/srep03379

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recently, gene editing with transcription activator-like effector nucleases (TALENs) has been used in the life sciences. TALENs can be easily customized to recognize a specific DNA sequence and efficiently introduce double-strand breaks at the targeted genomic locus. Subsequent non-homologous end-joining repair leads to targeted gene disruption by base insertion, deletion, or both. Here, to readily evaluate the efficacy of TALENs in Xenopus laevis embryos, we performed the targeted gene disruption of tyrosinase (tyr) and pax6 genes that are involved in pigmentation and eye formation, respectively. We constructed TALENs targeting tyr and pax6 and injected their mRNAs into fertilized eggs at the one-cell stage. Expectedly, introduction of tyr TALEN mRNA resulted in drastic loss of pigmentation with high efficiency. Similarly, for pax6, TALENs led to deformed eyes in the injected embryos. We confirmed mutations of the target alleles by restriction enzyme digestion and sequence analyses of genomic PCR products. Surprisingly, not only biallelic but also paralogous, gene disruption was observed. Our results demonstrate that targeted gene disruption by TALENs provides a method comparable to antisense morpholinos in analyzing gene function in Xenopus F0 embryos, but also applies beyond embryogenesis to any life stage. (C) 2013. Published by The Company of Biologists Ltd.

DOI： 10.1242/bio.20133855

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Transcription activator-like effector nucleases (TALENs) have recently arisen as effective tools for targeted genome engineering. Here, we report streamlined methods for the construction and evaluation of TALENs based on the 'Golden Gate TALEN and TAL Effector Kit' (Addgene). We diminished array vector requirements and increased assembly rates using six-module concatemerization. We altered the architecture of the native TALEN protein to increase nuclease activity and replaced the final destination vector with a mammalian expression/in vitro transcription vector bearing both CMV and T7 promoters. Using our methods, the whole process, from initiating construction to completing evaluation directly in mammalian cells, requires only 1 week. Furthermore, TALENs constructed in this manner may be directly applied to transfection of cultured cells or mRNA synthesis for use in animals and embryos. In this article, we show genomic modification of HEK293T cells, human induced pluripotent stem cells, Drosophila melanogaster, Danio rerio and Xenopus laevis, using custom-made TALENs constructed and evaluated with our protocol. Our methods are more time efficient compared with conventional yeast-based evaluation methods and provide a more accessible and effective protocol for the application of TALENs in various model organisms. © 2013 The Authors Genes to Cells © 2013 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd.

DOI： 10.1111/gtc.12037

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Lens growth involves the proliferation of epithelial cells, followed by their migration to the equator region and differentiation into secondary fiber cells. It is widely accepted that fibroblast growth factor (FGF) signaling is required for the differentiation of lens epithelial cells into crystallin-rich fibers, but this signaling is insufficient to induce full differentiation. To better understand lens development, investigatory and functional analyses of novel molecules are required. Here, we demonstrate that Equarin, which is a novel secreted molecule, was expressed exclusively in the lens equator region during chick lens development. Equarin upregulated the expression of fiber markers, as demonstrated using in ovo electroporation. In a primary lens cell culture, Equarin promoted the biochemical and morphological changes associated with the differentiation of lens epithelial cells to fibers. A loss-of-function analysis was performed using zinc-finger nucleases targeting the Equarin gene. Lens cell differentiation was markedly inhibited when endogenous Equarin was blocked, indicating that Equarin was essential for normal chick lens differentiation. Furthermore, biochemical analysis showed that Equarin directly bound to FGFs and heparan sulfate proteoglycan and thereby upregulated the expression of phospho-ERK1/2 (ERK-P) proteins, the downstream of the FGF signaling pathway, in vivo and in vitro. Conversely, the absence of endogenous Equarin clearly diminished FGF-induced fiber differentiation. Taken together, our results suggest that Equarin is involved as an FGF modulator in chick lens differentiation. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.ydbio.2012.05.029

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Hemimetabolous, or incompletely metamorphosing, insects are phylogenetically relatively basal and comprise many pests. However, the absence of a sophisticated genetic model system, or targeted gene-manipulation system, has limited research on hemimetabolous species. Here we use zinc-finger nuclease and transcription activator-like effector nuclease technologies to produce genetic knockouts in the hemimetabolous insect Gryllus bimaculatus. Following the microinjection of mRNAs encoding zinc-finger nucleases or transcription activator-like effector nucleases into cricket embryos, targeting of a transgene or endogenous gene results in sequence-specific mutations. Up to 48% of founder animals transmit disrupted gene alleles after zinc-finger nucleases microinjection compared with 17% after microinjection of transcription activator-like effector nucleases. Heterozygous offspring is selected using mutation detection assays that use a Surveyor (Cel-I) nuclease, and subsequent sibling crosses create homozygous knockout crickets. This approach is independent from a mutant phenotype or the genetic tractability of the organism of interest and can potentially be applied to manage insect pests using a non-transgenic strategy.

DOI： 10.1038/ncomms2020

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

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained similar to 1-kb homology arms and a 2A-histone H2B-GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.

DOI： 10.1073/pnas.1202768109

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Zinc-finger nucleases (ZFNs) are engineered nucleases that induce DNA double-strand breaks (DSBs) at target sequences. They have been used as tools for generating targeted mutations in the genomes of multiple organisms in both animals and plants. The DSB induced by ZFNs is repaired by non-homologous end joining (NHEJ) or by homologous recombination (HR) mechanisms. Non-homologous end joining induces some errors because it is independent of a reference DNA sequence. Through the NHEJ mechanism, ZFNs generate insertional or deletional mutations at the target sequence. We examined the usability, specificity and toxicity of ZFNs in the basal chordate Ciona intestinalis. As the target of ZFNs, we chose an enhanced green fluorescent protein (EGFP) gene artificially inserted in the C. intestinalis genome because this locus is neutral for the development and growth of C. intestinalis, and the efficiency of mutagenesis with ZFNs can thus be determined without any bias. We introduced EGFP-ZFN mRNAs into the embryos of an EGFP-transgenic line and observed the mutation frequency in the target site of EGFP . We also examined the effects of the EGFP-ZFNs at off-target sites resembling the EGFP target sequence in the C. intestinalis genome in order to examine the specificity of ZFNs. We further investigated the influence of ZFNs on embryogenesis, and showed that adequate amounts of ZFNs, which do not disrupt embryogenesis, can efficiently induce mutations on the on-target site with less effect on the off-target sites. This suggests that target mutagenesis with ZFNs will be a powerful technique in C. intestinalis.

DOI： 10.1111/j.1440-169X.2012.01355.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Zinc-finger nucleases (ZFNs) are artificial enzymes that create site-specific double-strand breaks and thereby induce targeted genome editing. Here, we demonstrated successful gene disruption in somatic and germ cells of medaka (Oryzias latipes) using ZFN to target exogenous EGFP genes. Embryos that were injected with an RNA sequence pair coding for ZFNs showed mosaic loss of green fluorescent protein fluorescence in skeletal muscle. A number of mutations that included both deletions and insertions were identified within the ZFN target site in each embryo, whereas no mutations were found at the non-targeted sites. In addition, ZFN-induced mutations were introduced in germ cells and efficiently transmitted to the next generation. The mutation frequency varied (6100%) in the germ cells from each founder, and a founder carried more than two types of mutation in germ cells. Our results have introduced the possibility of targeted gene disruption and reverse genetics in medaka.

DOI： 10.1111/j.1440-169X.2012.01357.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Sulfatases such as arylsulfatase and heparan sulfate 6-O-endosulfatase play important roles in morphogenesis during sea urchin development. For the activation of these sulfatases, C alpha-formylglycine formation by sulfatase modifying factor (Sumf) is required. In this study, to clarify the regulatory mechanisms for the activation of sulfatases during sea urchin development, we examined the expression and function of the Hemicentrotus pulcherrimus homologs of Sumf1 and Sumf2 (HpSumf1 and HpSumf2, respectively). Expression of HpSumf1 but not HpSumf2 mRNA was dynamically changed during early development. Functional analyses of recombinant HpSumf1 and HpSumf2 using HEK293T cells expressing mouse arylsulfatase A (ArsA) indicated that HpSumf1 and HpSumf2 were both able to activate mammalian ArsA. Knockdown of HpSumf1 using morpholino antisense oligonucleotides caused abnormal spicule formation in the sea urchin embryo. Injection of HpSumf2 mRNA had no effect on skeletogenesis, while injection of HpSumf1 mRNA induced severe supernumerary spicule formation. Taken together, these findings suggest that HpSumf1 is involved in the activation of sulfatases required for control of skeletogenesis.

DOI： 10.1007/s00427-011-0368-2

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

We showed that engineered zinc-finger nucleases (ZFNs), which consist of a zinc-finger DNA-binding array and a nuclease domain of the restriction enzyme FokI, can introduce mutations at a specific genomic site in the sea urchin embryo. Using bacterial one-hybrid screening with zinc-finger randomized libraries and a single-strand annealing assay in cultured cells, ZFNs targeting the sea urchin Hemicentrotus pulcherrimus homologue of HesC (HpHesC) were efficiently selected. Consistent with the phenotype observed in embryos injected with an antisense morpholino oligonucleotide against HpHesC, an increase in the primary mesenchyme cell population was observed in embryos injected with a pair of HpHesC ZFN mRNAs. In addition, sequence analysis of the mutations showed that deletions and insertions occurred at the HpHesC target site in the embryos injected with the HpHesC ZFN mRNAs. These results suggest that targeted gene disruption using ZFNs is feasible for the sea urchin embryo.

DOI： 10.1111/j.1365-2443.2010.01425.x

記述言語：英語   掲載種別：研究論文（学術雑誌）  

MicroRNAs are single-stranded RNA molecules with a length of 19-25 nucleotides, which play roles in various biological phenomena, including development, differentiation, apoptosis, by regulating target gene expression. Although the presence of microRNA molecules in sea urchin and the expression of genes involved in microRNA biogenesis during sea urchin development have been reported recently, the function of microRNA in sea urchin development remains to be elucidated. In this study, to understand the function of microRNA in the early development of sea urchin, we focused on Dicer, an essential enzyme for biosynthesis of mature microRNA. We determined the nucleotide sequence of cDNA for a Dicer homolog in the sea urchin, Hemicentrotus pulcherrimus, HpDcr, and found that functional domains of Dicer proteins are conserved in HpDcr. Analyses of its pattern of expression showed that HpDcr mRNA is expressed in embryos at all developmental stages analyzed, and seems to distribute asymmetrically at the morula and later stages. Knockdown of HpDcr resulted in anomalous morphogenesis, such as impairment of gastrulation and skeletogenesis at the mesenchyme blastula stage and later stages, and alteration of mRNA levels of cell type-specific genes. Thus, HpDcr plays important roles in morphogenesis in sea urchin embryos, suggesting that miRNA could be involved in the early development of sea urchin by regulating target gene expression.

DOI： 10.2108/zsj.27.477

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The nanos genes play important roles in the development of primordial germ cells in animal species. In the sea urchin, Hemicentrotus pulcherrimus, small micromere descendants specifically express HpNanos mRNA and this expression continues in the left coelomic pouch, which produces the major component of the adult rudiment. In this study, we showed that morpholino knockdown of HpNanos resulted in a delay of primary mesenchyme cell ingression and a decrease in the number of cells comprising the left coelomic pouch. Knockdown analysis in chimeras and whole embryos revealed the disappearance of small micromere descendants from the archenteron tip. Furthermore, the expression of HpNanos mRNA was induced in other cell lineages in the HpNanos-knockdown and micromere-deleted embryos. Taken together, our results suggest that HpNanos is involved in the inductive interaction of small micromere descendants with other cell lineages, and that HpNanos is required for the survival of small micromere descendants. Developmental Dynamics 238:2511-2521, 2009. (c) 2009 Wiley-Liss, Inc.

DOI： 10.1002/dvdy.22074

記述言語：英語   掲載種別：研究論文（学術雑誌）  

In sea urchin embryos, Notch signaling is required to segregate non-skeletogenic mesoderm from early endomesoderm, and is involved in endoderm development. To further investigate the role of Notch signaling in the endoderm cell lineage, we cloned a cDNA for the Hemicentrotus pulcherrimus ortholog of Suppressor of Hairless (HpSu(H)), which is a major mediator of the Notch signaling pathway, examined the expression during development and performed a functional analysis. HpSu(H) mRNA was ubiquitously expressed up to the unhatched blastula stage, and expression was exclusively detected in the vegetal plate region from the hatched blastula stage and then in the archenteron at the gastrula stage. Perturbation of HpSu(H) by injection of the dominant negative form of HpSu(H) (dn-HpSu(H)) mRNA into fertilized eggs led to the disappearance of secondary mesenchyme cells at the tip of the archenteron in the gastrula and pigment cells in the pluteus larva, confirming that Notch signaling is required for non-skeletogenic mesoderm specification. In addition, injection of relatively high amounts of dn-HpSu(H) mRNA caused a defect or atrophy of the foregut in the archenteron at the pluteus stage. This result strongly suggests that Notch signaling is involved in foregut development during sea urchin development.

DOI： 10.2108/zsj.26.686

記述言語：英語   掲載種別：研究論文（学術雑誌）  

For the efficient generation of transgenic sea urchins, we have adopted an I-SceI meganuclease-mediated transgenesis method. Several types of promoter-GFP gene constructs flanked by two I-SceI recognition sequences were co-injected with I-SceI into sea urchin fertilized eggs. Using cell-lineage-specific promoter constructs, the frequency of transgene expression was elevated, and their level of mozaicism was reduced. The addition of the Ars insulator sequence, which is known to block the enhancer activity and protect transgenes from position effects, led to a reduction in ectopic transgene expression and an elevation of transgene expression frequency in this I-SceI-mediated system. However, the magnitude of the effects of the Ars insulator was dependent upon the promoter constructs. QPCR analysis also showed that the Ars insulator increases the transgene copy number. These results suggest that the I-SceI-mediated method using the Ars insulator is advantageous for transgenesis in the sea urchin embryo.

DOI： 10.1002/dvdy.21690

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

In sea urchin development, micromere descendants play important roles in skeletogenesis and induction of gastrulation. We previously reported that the T-brain homolog of sea urchin Hemicentrotus pulcherrimus, HpTb expresses specifically in micromere descendants and is required for induction of gastrulation and skeletogenesis. Thus, HpTb is thought to play important roles in the function of micromere-lineage cells. To identify cis-regulatory regions responsible for spatio-temporal gene expression of HpTb, we isolated similar to 7 kb genomic region of HpTb gene and showed that GFP expression driven by this region exhibits the spatio-temporal pattern corresponding substantially to that of endogenous HpTb expression. Deletion of interspecifically conserved C2 and C4 regions resulted in an increase of ectopic expression. Mutations in Hairy family and Snail family consensus sequences in C1 and C2 regions also increased ectopic expression. Furthermore, we demonstrated that C4 region functions as enhancer, and that three Ets family consensus sequences are involved in this activity but not in spatial regulation. Therefore, we concluded that expression of HpTb gene is regulated by multiple cis-regulatory elements. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.mod.2007.10.009

DOI： 10.1101/2024.10.22.619557

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

DOI： 10.2352/ei.2024.36.15.coimg-161

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記述言語：その他  

担当ページ：総ページ数：207, 担当ページ数：62-72   記述言語：英語  

開催年月日： 2024年1月

記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：神戸, 理研BDR   国名：日本国  

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開催年月日： 2023年11月

記述言語：英語  

開催地：名古屋   国名：日本国  

開催年月日： 2023年11月

記述言語：英語   会議種別：口頭発表（招待・特別）  

開催地：名古屋   国名：日本国  

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その他リンク： https://www2.aeplan.co.jp/bsj2023/en-index.html

開催年月日： 2023年10月

記述言語：英語   会議種別：シンポジウム・ワークショップ パネル（指名）  

国名：日本国  

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開催年月日： 2023年10月

記述言語：日本語   会議種別：口頭発表（招待・特別）  

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開催年月日： 2023年9月

記述言語：英語   会議種別：シンポジウム・ワークショップ パネル（指名）  

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開催年月日： 2023年9月

記述言語：英語   会議種別：口頭発表（招待・特別）  

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開催年月日： 2023年6月

記述言語：日本語   会議種別：口頭発表（招待・特別）  

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開催年月日： 2023年5月

記述言語：日本語   会議種別：口頭発表（招待・特別）  

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開催年月日： 2023年3月

記述言語：日本語   会議種別：口頭発表（招待・特別）  

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開催年月日： 2022年12月

記述言語：日本語  

国名：その他  

開催年月日： 2022年12月

記述言語：日本語   会議種別：口頭発表（招待・特別）  

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開催年月日： 2021年2月

会議種別：口頭発表（招待・特別）  

開催地：オンライン  

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記述言語：英語  

国名：日本国  

Unveiling transcriptional dynamics in pluripotent stem cells through imaging analysis

記述言語：英語  

国名：その他  

TALEN-mediated single-base-pair editing reveals the functional significance of an intergenic single nucleotide variant

記述言語：英語  

国名：その他  

TALEN-mediated single-base-pair editing in PCS syndrome

記述言語：英語  

国名：その他  

Stochastic promoter activation affects gene expression variability in murine embryonic stem cells

記述言語：日本語  

国名：その他  

記述言語：英語  

国名：その他  

Simultaneous live-imaging of gene position and its transcriptional activity in mouse embryonic stem cells

記述言語：日本語  

開催地：熊本大学   国名：その他  

記述言語：英語  

国名：その他  

Simultaneous Live Imaging of the Transcription and Nuclear Position of Specific Genes

記述言語：日本語  

開催地：広島大学   国名：その他  

記述言語：英語  

国名：その他  

Simultaneous live imaging of the transcription and nuclear position of specific genes

記述言語：日本語  

国名：その他  

記述言語：英語  

国名：その他  

Simultaneous live imaging of a specific gene's transcription and dynamics.

記述言語：英語  

国名：その他  

Active nuclear dynamics promotes heterochromatin clustering

記述言語：英語  

国名：その他  

Relationship between nuclear dynamics and gene expression noise in pluripotent stem cell

記述言語：英語  

国名：その他  

A challenging interdisciplinary approach to elucidate a mystery of remodeling process in nuclear architecture (Part II: Experiment)

記述言語：英語  

国名：その他  

Single Base Pair Genome-Editing Technology and Its Future Applications

記述言語：英語  

国名：その他  

Dynamic nuclear deformation induces nuclear architecture remodeling

記述言語：日本語  

国名：その他  

記述言語：日本語  

国名：その他  

記述言語：日本語  

国名：その他  

記述言語：英語  

国名：その他  

Relationship between kinetics of higher-order genomic structure and transcriptional activity

記述言語：日本語  

開催地：神戸   国名：その他  

記述言語：日本語  

開催地：かでる2・7（北海道⽴道⺠活動センター）   国名：その他  

記述言語：日本語  

開催地：大阪国際会議場   国名：その他  

記述言語：日本語  

国名：その他  

記述言語：日本語  

国名：その他  

記述言語：日本語  

国名：その他  

記述言語：日本語  

国名：その他  

記述言語：英語  

開催地：東京   国名：その他  

記述言語：英語  

国名：その他  

Transcriptional bursting induces gene expression heterogeneity in mouse embryonic stem cells

記述言語：英語  

国名：その他  

Transcriptional bursting induces gene expression heterogeneity in mES cells

記述言語：その他  

国名：その他  

記述言語：その他  

開催地：オンライン   国名：その他  

記述言語：その他  

開催地：オンライン   国名：その他  

記述言語：その他  

国名：その他  

Single-gene imaging reveals the relationship between the transcriptional activity of single genes and the formation of transcriptional regulatory factor clusters

記述言語：その他  

国名：その他  

Relationship between higher-order genome structural dynamics and transcriptional dynamics

記述言語：その他  

開催地：オンライン   国名：その他  

記述言語：その他  

国名：その他  

Genome-wide kinetic properties of transcriptional bursting revealed by single cell analysis

記述言語：その他  

国名：その他  

STREAMING-tag system: A novel technology to analyze the spatiotemporalrelationship between transcriptional regulators and transcriptionaldynamics at the single gene level

記述言語：その他  

開催地：郡山   国名：その他  

記述言語：その他  

開催地：三島   国名：その他  

記述言語：その他  

開催地：Online   国名：その他  

記述言語：その他  

開催地：国立遺伝学研究所   国名：その他  

記述言語：その他  

国名：その他  

Revealing regulatory mechanisms of transcriptional dynamics by single-gene imaging system

記述言語：その他  

開催地：広島大学東広島キャンパス 学士会館   国名：その他  

記述言語：その他  

開催地：幕張メッセ、千葉   国名：その他  

記述言語：その他  

国名：その他  

Transcriptional dynamics regulated by non-genomic codes

記述言語：その他  

国名：その他  

Localization of transcriptional regulator condensates in transcriptional bursting revealed by STREAMING-tag system

記述言語：英語  

開催地：オンライン   国名：その他  

開催地：Online  

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開催地：国立遺伝学研究所  

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開催地：広島大学東広島キャンパス 学士会館  

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開催地：郡山  

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開催地：幕張メッセ、千葉  

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開催地：三島  

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記述言語：英語   会議種別：公開講演，セミナー，チュートリアル，講習，講義等  

開催地：RIKEN IMS   国名：日本国  

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開催地：Ito Hall, The University of Tokyo  

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記述言語：英語   会議種別：口頭発表（招待・特別）  

開催地：つくば市   国名：日本国  

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開催地：Online  

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開催地：Sydney, Australia  

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記述言語：英語   会議種別：シンポジウム・ワークショップ パネル（指名）  

開催地：オンライン  

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開催地：Zoom online  

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記述言語：日本語  

CRISPR-Cas9システムを利用したゲノム編集技術は、基礎研究利用に留まらず、医療応用や、有用畜産物の作出など、広く革新をもたらしている。一方で、DNAを切断しないCas9変異体を用いることで、ゲノム編集とは異なる応用も可能である。その1つが、CRISPR-Cas9システムを利用した特定遺伝子座のライブイメージングである。本稿では、特定ゲノム領域の可視化の意義、CRISPR-Cas9による生細胞イメージングのツールボックス、さらに本技術を利用した応用研究を紹介し、今後の展望も踏まえて議論する。(著者抄録)

記述言語：日本語  

DOI： 10.2142/biophys.61.171

記述言語：日本語  

光学顕微鏡が普及し、肉眼では見えない細胞などの微細構造を観察できるようになり、多くの新しい生命現象の発見を支えてきた。とくに蛍光顕微鏡とその関連技術は、培養細胞のみならず、厚い組織切片、オルガノイド、生体などにおいて、特定分子の細胞内"動態"や発現量の"経時的変化"の測定を可能にし、創薬や病因同定などの医学分野にも深く関係している。本稿では、蛍光の原理および、1細胞レベルでのライブイメージングに用いられる蛍光顕微鏡、およびその派生技術である共焦点顕微鏡、多光子励起顕微鏡、ライトシート顕微鏡について解説する。さらに、1細胞レベルでの動態解析の重要性、蛍光ラベル化法、疾患オルガノイドを例にした医療への応用について解説する。(著者抄録)

記述言語：日本語  

記述言語：日本語  

DOI： 10.11477/mf.2425200621

記述言語：日本語  

記述言語：英語  

DOI： 10.1111/gtc.12037

記述言語：日本語  

記述言語：英語  

記述言語：日本語  

記述言語：日本語  

記述言語：日本語  

記述言語：英語  

DOI： 10.1007/978-4-431-55227-7_1

種別：学会・研究会等 

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種別：学会・研究会等 

種別：大会・シンポジウム等 

種別：審査・学術的助言 

種別：大会・シンポジウム等 

種別：大会・シンポジウム等 

種別：大会・シンポジウム等 

対象： 社会人・一般,　学術団体,　企業,　市民団体,　行政機関

種別：出前授業

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対象： 社会人・一般,　学術団体,　企業,　市民団体,　行政機関

種別：出前授業

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対象： 社会人・一般,　学術団体,　企業,　市民団体,　行政機関

種別：インターネット

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対象： 社会人・一般,　学術団体,　企業,　市民団体,　行政機関

種別：新聞・雑誌

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対象： 社会人・一般,　学術団体,　企業,　市民団体,　行政機関

広島大が標的遺伝子の細胞内位置と活性を同時に可視化するROLEX技術、CRISPR/dCas9を活用

Simultaneous live imaging of a specific gene's transcription and dynamics

Simultaneous live imaging of a specific gene's transcription and dynamics

マウス胚性幹細胞における確率的なプロモーター活性化がNanog発現のばらつきに影響を与える

【山本研ゲノム編集アップデイト(3)】Wageningen大、DNAガイドを介した新規DNA切断現象を原核生物で発見

広島大と理研が人工ヌクレアーゼZFNで遺伝子挿入、ウニ初期胚1細胞の遺伝子発現を可視化