業績リスト

@article{10.1101/2023.10.17.561810,
title = {Precise immunofluorescence canceling for highly multiplexed imaging capturing specific cell state},
author = {Kosuke Tomimatsu and Takeru Fujii and Ryoma Bise and Kazufumi Hosoda and Yosuke Taniguchi and Hiroshi Ochiai and Hiroaki Ohishi and Kanta Ando and Ryoma Minami and Kaori Tanaka and Taro Tachibana and Seiichi Mori and Akihito Harada and Kazumitsu Maehara and Masao Nagasaki and Seiichi Uchida and Hiroshi Kimura and Masashi Narita and Yasuyuki Ohkawa},
doi = {10.1101/2023.10.17.561810},
year  = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {bioRxiv},
pages = {2023.10.17.561810},
abstract = {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 a comprehensive acquisition of spatial protein information from cells. However, the specificity of antibodies is still compromised when visualizing activated signals. Here, we developed Precise Emission Canceling Antibodies (PECAbs) that have cleavable fluorescent labeling. PECAbs enable high-specificity sequential imaging using hundreds of antibodies, allowing for 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{10.1101/2023.11.27.568629,
title = {Transcription-coupled changes in higher-order genomic structure and transcription hub viscosity prolong enhancer-promoter connectivity},
author = {Hiroaki Ohishi and Soya Shinkai and Hitoshi Owada and Takeru Fujii and Kazufumi Hosoda and Shuichi Onami and Takashi Yamamoto and Yasuyuki Ohkawa and Hiroshi Ochiai},
doi = {10.1101/2023.11.27.568629},
year  = {2023},
date = {2023-01-01},
urldate = {2023-01-01},
journal = {bioRxiv},
pages = {2023.11.27.568629},
abstract = {The orchestration of our genes heavily relies on a coordinated communication between enhancers and promoters, yet how this dynamic interplay remains elusive while transcription is active. Here, we investigated enhancer-promoter (E-P) interactions and relative to transcriptional bursting in mouse embryonic stem cells using sequential DNA/RNA/immunofluorescence (IF)-FISH analyses and computational simulations. Our data reveal that the active state of specific genes is characterized by higher-order genomic structures and local condensates of transcriptional regulatory factors, leading to an elevation in local viscosity that highly stabilizes the duration of E-P interactions. Our study underscores the pivotal role of viscosity in transcriptional dynamics and paves the way for a more nuanced understanding of gene-specific regulatory mechanisms.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{pmid36539402,
title = {STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity},
author = {Hiroaki Ohishi and Seiru Shimada and Satoshi Uchino and Jieru Li and Yuko Sato and Manabu Shintani and Hitoshi Owada and Yasuyuki Ohkawa and Alexandros Pertsinidis and Takashi Yamamoto and Hiroshi Kimura and Hiroshi Ochiai},
doi = {10.1038/s41467-022-35286-2},
issn = {2041-1723},
year  = {2022},
date = {2022-12-01},
journal = {Nat Commun},
volume = {13},
number = {1},
pages = {7672},
abstract = {Transcription is a dynamic process. To detect the dynamic relationship among protein clusters of RNA polymerase II and coactivators, gene loci, and transcriptional activity, we insert an MS2 repeat, a TetO repeat, and inteins with a selection marker just downstream of the transcription start site. By optimizing the individual elements, we develop the Spliced TetO REpeAt, MS2 repeat, and INtein sandwiched reporter Gene tag (STREAMING-tag) system. Clusters of RNA polymerase II and BRD4 are observed proximal to the transcription start site of Nanog when the gene is transcribed in mouse embryonic stem cells. In contrast, clusters of MED19 and MED22 tend to be located near the transcription start site, even without transcription activity. Thus, the STREAMING-tag system reveals the spatiotemporal relationships between transcriptional activity and protein clusters near the gene. This powerful tool is useful for quantitatively understanding transcriptional regulation in living cells.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{10.1371/journal.pone.0264965,
title = {iPSC reprogramming-mediated aneuploidy correction in autosomal trisomy syndromes},
author = {Silvia Natsuko Akutsu and Tatsuo Miyamoto and Daiju Oba and Keita Tomioka and Hiroshi Ochiai and Hirofumi Ohashi and Shinya Matsuura},
doi = {10.1371/journal.pone.0264965},
year  = {2022},
date = {2022-01-01},
journal = {PLoS ONE},
volume = {17},
number = {3},
pages = {e0264965},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{10.26508/lsa.202101045,
title = {Ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly},
author = {Haruka Matsumori and Kenji Watanabe and Hiroaki Tachiwana and Tomoko Fujita and Yuma Ito and Makio Tokunaga and Kumiko Sakata-Sogawa and Hiroko Osakada and Tokuko Haraguchi and Akinori Awazu and Hiroshi Ochiai and Yuka Sakata and Koji Ochiai and Tsutomu Toki and Etsuro Ito and Ilya G Goldberg and Kazuaki Tokunaga and Mitsuyoshi Nakao and Noriko Saitoh},
doi = {10.26508/lsa.202101045},
issn = {2575-1077},
year  = {2022},
date = {2022-01-01},
journal = {Life Science Alliance},
volume = {5},
number = {7},
abstract = {The nucleolus is the site of ribosome assembly and formed through liquid\textendashliquid 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\textendashliquid 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\textendashBlackfan 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{10.1016/j.ggedit.2022.100022,
title = {Facilitating genome function understanding using genome editing dependent bioimaging techniques},
author = {Hiroshi Ochiai},
doi = {10.1016/j.ggedit.2022.100022},
issn = {2666-3880},
year  = {2022},
date = {2022-01-01},
urldate = {2022-01-01},
journal = {Gene and Genome Editing},
volume = {5},
pages = {100022},
abstract = {Genomic DNA is highly folded and is stored in the nucleus. In multicellular organisms, genomic DNA exhibits cell type-specific higher-order structures, including specific enhancer\textendashpromoter interactions, which have recently been shown to be relevant for cell type-specific regulation of gene expression. However, when the distances between specific enhancers and promoters were measured, cell-to-cell heterogeneity was unexpectedly large and, in some cases, did not correlate with the transcriptional state. These phenomena can be revealed by simultaneously visualizing specific genomic regions and the biological phenomena of interest. In this mini-review, I introduce methodologies for visualizing specific genomic DNA regions and provide detailed examples of how these techniques are being used to elucidate the mechanisms of transcriptional regulation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{10.1101/2021.11.29.470307,
title = {Distinctive nuclear zone for RAD51-mediated homologous recombinational DNA repair},
author = {Yasunori Horikoshi and Hiroki Shima and Jiying Sun and Wataru Kobayashi and Volker J. Schmid and Hiroshi Ochiai and Lin Shi and Atsuhiko Fukuto and Yasuha Kinugasa and Hitoshi Kurumizaka and Tsuyoshi Ikura and Yolanda Markaki and Shin-ichi Tate and Kazuhiko Igarashi and Thomas Cremer and Satoshi Tashiro},
doi = {10.1101/2021.11.29.470307},
year  = {2021},
date = {2021-01-01},
journal = {bioRxiv},
pages = {2021.11.29.470307},
abstract = {Genome-based functions are inseparable from the dynamic higher-order architecture of the cell nucleus. In this context, the repair of DNA damage is coordinated by precise spatiotemporal controls that target and regulate the repair machinery required to maintain genome integrity. However, the mechanisms that pair damaged DNA with intact template for repair by homologous recombination (HR) without illegitimate recombination remain unclear. This report highlights the intimate relationship between nuclear architecture and HR in mammalian cells. RAD51, the key recombinase of HR, forms spherical foci in S/G2 phases spontaneously. Using super-resolution microscopy, we show that following induction of DNA double-strand breaks RAD51 foci at damaged sites elongate to bridge between intact and damaged sister chromatids; this assembly occurs within bundle-shaped distinctive nuclear zones, requires interactions of RAD51 with various factors, and precedes ATP-dependent events involved the recombination of intact and damaged DNA. We observed a time-dependent transfer of single-stranded DNA overhangs, generated during HR, into such zones. Our observations suggest that RAD51-mediated homologous pairing during HR takes place within the distinctive nuclear zones to execute appropriate recombination.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Ochiai:2020gy,
title = {Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells},
author = {Hiroshi Ochiai and Tetsutaro Hayashi and Mana Umeda and Mika Yoshimura and Akihito Harada and Yukiko Shimizu and Kenta Nakano and Noriko Saitoh and Zhe Liu and Takashi Yamamoto and Tadashi Okamura and Yasuyuki Ohkawa and Hiroshi Kimura and Itoshi Nikaido},
url = {https://advances.sciencemag.org/lookup/doi/10.1126/sciadv.aaz6699},
doi = {10.1126/sciadv.aaz6699},
year  = {2020},
date = {2020-01-01},
journal = {Science Advances},
volume = {6},
number = {25},
pages = {eaaz6699},
abstract = {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\textendashbinding proteins, including the polycomb repressive complex 2 and transcription elongation factors. Furthermore, large-scale CRISPR-Cas9\textendashbased 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Miyamoto:2020dk,
title = {Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome},
author = {Tatsuo Miyamoto and Kosuke Hosoba and Takeshi Itabashi and Atsuko H Iwane and Silvia Natsuko Akutsu and Hiroshi Ochiai and Yumiko Saito and Takashi Yamamoto and Shinya Matsuura},
url = {https://www.embopress.org/doi/full/10.15252/embj.2019103499},
doi = {10.15252/embj.2019103499},
issn = {0261-4189},
year  = {2020},
date = {2020-01-01},
journal = {The EMBO Journal},
volume = {39},
number = {12},
pages = {e103499},
abstract = {Primary cilia are antenna‐like organelles on the surface of most mammalian cells that receive sonic hedgehog (Shh) signaling in embryogenesis and carcinogenesis. Cellular cholesterol functions as a direct activator of a seven‐transmembrane oncoprotein called Smoothened (Smo) and thereby induces Smo accumulation on the ciliary membrane where it transduces the Shh signal. However, how cholesterol is supplied to the ciliary membrane remains unclear. Here, we report that peroxisomes are essential for the transport of cholesterol into the ciliary membrane. Zellweger syndrome (ZS) is a peroxisome‐deficient hereditary disorder with several ciliopathy‐related features and cells from these patients showed a reduced cholesterol level in the ciliary membrane. Reverse genetics approaches revealed that the GTP exchange factor Rabin8, the Rab GTPase Rab10, and the microtubule minus‐end‐directed kinesin KIFC3 form a peroxisome‐associated complex to control the movement of peroxisomes along microtubules, enabling communication between peroxisomes and ciliary pocket membranes. Our findings suggest that insufficient ciliary cholesterol levels may underlie ciliopathies.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Shinkai:2020dh,
title = {PHi-C: deciphering Hi-C data into polymer dynamics},
author = {Soya Shinkai and Masaki Nakagawa and Takeshi Sugawara and Yuichi Togashi and Hiroshi Ochiai and Ryuichiro Nakato and Yuichi Taniguchi and Shuichi Onami},
url = {https://academic.oup.com/nargab/article/doi/10.1093/nargab/lqaa020/5813971},
doi = {10.1093/nargab/lqaa020},
year  = {2020},
date = {2020-01-01},
journal = {NAR Genomics and Bioinformatics},
volume = {2},
number = {2},
pages = {lqaa020},
abstract = {Genomes are spatiotemporally organized within the cell nucleus. Genome-wide chromosome conformation capture (Hi-C) technologies have uncovered the 3D genome organization. Furthermore, live-cell imaging experiments have revealed that genomes are functional in 4D. Although computational modeling methods can convert 2D Hi-C data into population-averaged static 3D genome models, exploring 4D genome nature based on 2D Hi-C data remains lacking. Here, we describe a 4D simulation method, PHi-C (polymer dynamics deciphered from Hi-C data), that depicts 4D genome features from 2D Hi-C data by polymer modeling. PHi-C allows users to interpret 2D Hi-C data as physical interaction parameters within single chromosomes. The physical interaction parameters can then be used in the simulations and analyses to demonstrate dynamic characteristics of genomic loci and chromosomes as observed in live-cell imaging experiments. PHi-C is available at https://github.com/soyashinkai/PHi-C.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Li:2020da,
title = {Single-gene imaging links genome topology, promoter-enhancer communication and transcription control},
author = {Jieru Li and Angela Hsu and Yujing Hua and Guanshi Wang and Lingling Cheng and Hiroshi Ochiai and Takashi Yamamoto and Alexandros Pertsinidis},
url = {http://dx.doi.org/10.1038/s41594-020-0493-6},
doi = {10.1038/s41594-020-0493-6},
issn = {1545-9993},
year  = {2020},
date = {2020-01-01},
journal = {Nature structural \& molecular biology},
volume = {27},
number = {11},
pages = {1032--1040},
abstract = {Transcription activation by distal enhancers is essential for cell-fate specification and maintenance of cellular identities. How long-range gene regulation is physically achieved, especially within complex regulatory landscapes of non-binary enhancer-promoter configurations, remains elusive. Recent nanoscopy advances quantitatively linked promoter kinetics and textbackslashtextasciitilde100\textendash200 nm-sized clusters of enhancer-associated regulatory factors (RFs) at important developmental genes. Here, we further dissect mechanisms of RF clustering and transcription activation in mouse embryonic stem cells. RF recruitment into clusters involves specific molecular recognition of cognate DNA and chromatin binding sites, suggesting underlying cis-element clustering. Strikingly, imaging tagged genomic loci, with ≤1 kilobase and ≈20 nanometer precision, in live cells, reveals distal enhancer clusters over the extended locus in frequent close proximity to target genes - within RF clustering distances. These high-interaction-frequency enhancer cluster “super-clusters” create nano-environments wherein clustered RFs activate target genes, providing a structural framework for relating genome organization, focal RF accumulation and transcription activation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Anonymous:2019gt,
title = {Role of dynamic nuclear deformation on genomic architecture reorganization},
author = {Sungrim Seirin-Lee and Fumitaka Osakada and Junichi Takeda and Satoshi Tashiro and Ryo Kobayashi and Takashi Yamamoto and Hiroshi Ochiai},
editor = {Alexandre V Morozov},
url = {http://dx.plos.org/10.1371/journal.pcbi.1007289},
doi = {10.1371/journal.pcbi.1007289},
issn = {1553-734X},
year  = {2019},
date = {2019-01-01},
journal = {PLoS Computational Biology},
volume = {15},
number = {9},
pages = {e1007289},
abstract = {Higher-order genomic architecture varies according to cell type and changes dramatically during differentiation. One of the remarkable examples of spatial genomic reorganization is the rod photoreceptor cell differentiation in nocturnal mammals. The inverted nuclear architecture found in adult mouse rod cells is formed through the reorganization of the conventional architecture during terminal differentiation. However, the mechanisms underlying these changes remain largely unknown. Here, we found that the dynamic deformation of nuclei via actomyosin-mediated contractility contributes to chromocenter clustering and promotes genomic architecture reorganization during differentiation by conducting an in cellulo experiment coupled with phase-field modeling. Similar patterns of dynamic deformation of the nucleus and a concomitant migration of the nuclear content were also observed in rod cells derived from the developing mouse retina. These results indicate that the common phenomenon of dynamic nuclear deformation, which accompanies dynamic cell behavior, can be a universal mechanism for spatiotemporal genomic reorganization.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Li:2019bo,
title = {Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells},
author = {Jieru Li and Ankun Dong and Kamola Saydaminova and Hill Chang and Guanshi Wang and Hiroshi Ochiai and Takashi Yamamoto and Alexandros Pertsinidis},
url = {https://linkinghub.elsevier.com/retrieve/pii/S0092867419305574},
doi = {10.1016/j.cell.2019.05.029},
issn = {0092-8674},
year  = {2019},
date = {2019-01-01},
journal = {Cell},
volume = {178},
number = {2},
pages = {491--506.e28},
abstract = {Transforming the vast knowledge from genetics, biochemistry, and structural biology into detailed molecular descriptions of biological processes inside cells remains a major challenge\textemdashone in sore need of better imaging technologies. For example, transcription involves the complex interplay between RNA polymerase II (Pol II), regulatory factors (RFs), and chromatin, but visualizing these dynamic molecular transactions in their native intracellular milieu remains elusive. Here, we zoom into single tagged genes using nanoscopy techniques, including an active target-locking, ultra-sensitive system that enables single-molecule detection in addressable sub-diffraction volumes, within crowded intracellular environments. We image, track, and quantify Pol II with single-molecule resolution, unveiling its dynamics during the transcription cycle. Further probing multiple functionally linked events\textemdashRF-chromatin interactions, Pol II dynamics, and nascent transcription kinetics\textemdashreveals detailed operational parameters of gene-regulatory mechanisms hitherto-unseen in vivo. Our approach sets the stage for single-molecule studies of complex molecular processes in live cells.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Matsushita:2017gg,
title = {Dynamic changes in the interchromosomal interaction of early histone gene loci during development of sea urchin},
author = {Masaya Matsushita and Hiroshi Ochiai and Ken-ichi T. Suzuki and Sayaka Hayashi and Takashi Yamamoto and Akinori Awazu and Naoaki Sakamoto},
url = {http://jcs.biologists.org/lookup/doi/10.1242/jcs.206862},
doi = {10.1242/jcs.206862},
issn = {0021-9533},
year  = {2017},
date = {2017-01-01},
journal = {Journal of Cell Science},
volume = {130},
number = {24},
pages = {4097--4107},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Ishihara:2017ha,
title = {Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content},
author = {Satoru Ishihara and Naoe Kotomura and Naoki Yamamoto and Hiroshi Ochiai},
url = {http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed\&id=28502712\&retmode=ref\&cmd=prlinks},
doi = {10.1016/j.ab.2017.05.011},
issn = {0003-2697},
year  = {2017},
date = {2017-01-01},
journal = {Analytical Biochemistry},
volume = {531},
pages = {37--44},
abstract = { 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Matsuura:2016gt,
title = {Analysis of individual differences in radiosensitivity using genome editing.},
author = {Shinya Matsuura and Ekaterina Royba and Silvia N Akutsu and Hiromi Yanagihara and Hiroshi Ochiai and Yoshiki Kudo and Satoshi Tashiro and Tatsuo Miyamoto},
url = {http://eutils.ncbi.nlm.nih.gov/entrez/eutils/elink.fcgi?dbfrom=pubmed\&id=27012844\&retmode=ref\&cmd=prlinks},
doi = {10.1177/0146645316633941},
year  = {2016},
date = {2016-06-01},
journal = {Ann ICRP},
volume = {45},
number = {1 Suppl},
pages = {290--296},
abstract = {Current standards for radiological protection of the public have been uniformly established. However, individual differences in radiosensitivity are suggested to exist in human populations, which could be caused by nucleotide variants of DNA repair genes. In order to verify if such genetic variants are responsible for individual differences in radiosensitivity, they could be introduced into cultured human cells for evaluation. This strategy would make it possible to analyse the effect of candidate nucleotide variants on individual radiosensitivity, independent of the diverse genetic background. However, efficient gene targeting in cultured human cells is difficult due to the low frequency of homologous recombination (HR) repair. The development of artificial nucleases has enabled efficient HR-mediated genome editing to be performed in cultured human cells. A novel genome editing strategy, 'transcription activator-like effector nuclease (TALEN)-mediated two-step single base pair editing', has been developed, and this was used to introduce a nucleotide variant associated with a chromosomal instability syndrome bi-allelically into cultured human cells to demonstrate that it is the causative mutation. It is proposed that this editing technique will be useful to investigate individual radiosensitivity.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Ochiai:2015bt,
title = {Single-Base Pair Genome Editing in Human Cells by Using Site-Specific Endonucleases},
author = {Hiroshi Ochiai},
url = {http://www.mdpi.com/1422-0067/16/9/21128/htm},
doi = {10.3390/ijms160921128},
year  = {2015},
date = {2015-09-01},
journal = {Int J Mol Sci},
volume = {16},
number = {9},
pages = {21128--21137},
publisher = {Multidisciplinary Digital Publishing Institute},
abstract = {Genome-wide association studies have identified numerous single-nucleotide polymorphisms (SNPs) associated with human diseases or phenotypes. However, causal relationships between most SNPs and the associated disease have not been established, owing to technical challenges such as unavailability of suitable cell lines. Recently, efficient editing of a single base pair in the genome was achieved using programmable site-specific nucleases. This technique enables experimental confirmation of the causality between SNPs and disease, and is potentially valuable in clinical applications. In this review, I introduce the molecular basis and describe examples of single-base pair editing in human cells. I also discuss the challenges associated with the technique, as well as possible solutions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Ochiai:2015cy,
title = {Simultaneous live imaging of the transcription and nuclear position of specific genes},
author = {Hiroshi Ochiai and Takeshi Sugawara and Takashi Yamamoto},
url = {http://nar.oxfordjournals.org/lookup/doi/10.1093/nar/gkv624},
doi = {10.1093/nar/gkv624},
issn = {0305-1048},
year  = {2015},
date = {2015-01-01},
journal = {Nucleic Acids Research},
volume = {43},
number = {19},
pages = {e127--e127},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Miyamoto:2015fo,
title = {The Microtubule-Depolymerizing Activity of a Mitotic Kinesin Protein KIF2A Drives Primary Cilia Disassembly Coupled with Cell Proliferation},
author = {Tatsuo Miyamoto and Kosuke Hosoba and Hiroshi Ochiai and Ekaterina Royba and Hideki Izumi and Tetsushi Sakuma and Takashi Yamamoto and Brian David Dynlacht and Shinya Matsuura},
url = {http://linkinghub.elsevier.com/retrieve/pii/S2211124715000042},
doi = {10.1016/j.celrep.2015.01.003},
issn = {2211-1247},
year  = {2015},
date = {2015-01-01},
journal = {Cell Reports},
volume = {10},
number = {5},
pages = {664--673},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Ochiai:2014hv,
title = {Stochastic promoter activation affects Nanog expression variability in mouse embryonic stem cells},
author = {Hiroshi Ochiai and Takeshi Sugawara and Tetsushi Sakuma and Takashi Yamamoto},
url = {http://www.nature.com/doifinder/10.1038/srep07125},
doi = {10.1038/srep07125},
year  = {2014},
date = {2014-01-01},
journal = {Scientific Reports},
volume = {4},
number = {1},
pages = {7125},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Ochiai:2014era,
title = {TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUB1B as causative of PCS (MVA) syndrome},
author = {Hiroshi Ochiai and Tatsuo Miyamoto and Akinori Kanai and Kosuke Hosoba and Tetsushi Sakuma and Yoshiki Kudo and Keiko Asami and Atsushi Ogawa and Akihiro Watanabe and Tadashi Kajii and Takashi Yamamoto and Shinya Matsuura},
url = {http://www.pnas.org/cgi/doi/10.1073/pnas.1317008111},
doi = {10.1073/pnas.1317008111},
issn = {0027-8424},
year  = {2014},
date = {2014-01-01},
journal = {Proceedings of the National Academy of Sciences},
volume = {111},
number = {4},
pages = {1461--1466},
abstract = {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 monoallelic mutations 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\textendashmediated 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Sakuma:2013ix,
title = {Efficient TALEN construction and evaluation methods for human cell and animal applications},
author = {Tetsushi Sakuma and Sayaka Hosoi and Knut Woltjen and Ken‐ichi Suzuki and Keiko Kashiwagi and Housei Wada and Hiroshi Ochiai and Tatsuo Miyamoto and Narudo Kawai and Yasunori Sasakura and Shinya Matsuura and Yasushi Okada and Atsuo Kawahara and Shigeo Hayashi and Takashi Yamamoto},
url = {http://doi.wiley.com/10.1111/gtc.12037},
doi = {10.1111/gtc.12037},
issn = {1365-2443},
year  = {2013},
date = {2013-01-01},
journal = {Genes to Cells},
volume = {18},
number = {4},
pages = {315--326},
abstract = {Transcription activator\textendashlike 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Suzuki:2013ex,
title = {High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos},
author = {Ken-ichi T. Suzuki and Yukiko Isoyama and Keiko Kashiwagi and Tetsushi Sakuma and Hiroshi Ochiai and Naoaki Sakamoto and Nobuaki Furuno and Akihiko Kashiwagi and Takashi Yamamoto},
url = {http://bio.biologists.org/content/2/5/448.full},
doi = {10.1242/bio.20133855},
issn = {2046-6390},
year  = {2013},
date = {2013-01-01},
journal = {Biology Open},
volume = {2},
number = {5},
pages = {448--452},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Sakuma:2013bo,
title = {Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity},
author = {Tetsushi Sakuma and Hiroshi Ochiai and Takehito Kaneko and Tomoji Mashimo and Daisuke Tokumasu and Yuto Sakane and Ken-ichi Suzuki and Tatsuo Miyamoto and Naoaki Sakamoto and Shinya Matsuura and Takashi Yamamoto},
url = {http://www.nature.com/articles/srep03379},
doi = {10.1038/srep03379},
year  = {2013},
date = {2013-01-01},
journal = {Scientific Reports},
volume = {3},
number = {1},
pages = {3379},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Anonymous:2012gl,
title = {Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc‐finger nucleases},
author = {Narudo Kawai and Hiroshi Ochiai and Tetsushi Sakuma and Lixy Yamada and Hitoshi Sawada and Takashi Yamamoto and Yasunori Sasakura},
url = {http://doi.wiley.com/10.1111/j.1440-169X.2012.01355.x},
doi = {10.1111/j.1440-169x.2012.01355.x},
issn = {1440-169X},
year  = {2012},
date = {2012-01-01},
journal = {Development, Growth \& Differentiation},
volume = {54},
number = {5},
pages = {535--545},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Song:2012dq,
title = {Equarin is involved as an FGF signaling modulator in chick lens differentiation},
author = {Xiaohong Song and Yuya Sato and Athary Felemban and Ayako Ito and Mahmud Hossain and Hiroshi Ochiai and Takashi Yamamoto and Kiyotoshi Sekiguchi and Hideaki Tanaka and Kunimasa Ohta},
url = {http://pubmed.gov/22659080},
doi = {10.1016/j.ydbio.2012.05.029},
issn = {0012-1606},
year  = {2012},
date = {2012-01-01},
journal = {Developmental Biology},
volume = {368},
number = {1},
pages = {109--117},
abstract = { 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Watanabe:2012ia,
title = {Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases},
author = {Takahito Watanabe and Hiroshi Ochiai and Tetsushi Sakuma and Hadley W. Horch and Naoya Hamaguchi and Taro Nakamura and Tetsuya Bando and Hideyo Ohuchi and Takashi Yamamoto and Sumihare Noji and Taro Mito},
url = {http://www.nature.com/doifinder/10.1038/ncomms2020},
doi = {10.1038/ncomms2020},
year  = {2012},
date = {2012-01-01},
journal = {Nature Communications},
volume = {3},
number = {1},
pages = {1017},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Anonymous:2012jx,
title = {Targeted disruption of exogenous EGFP gene in medaka using zinc‐finger nucleases},
author = {Satoshi Ansai and Hiroshi Ochiai and Yuta Kanie and Yasuhiro Kamei and Yuki Gou and Takeshi Kitano and Takashi Yamamoto and Masato Kinoshita},
url = {http://doi.wiley.com/10.1111/j.1440-169X.2012.01357.x},
doi = {10.1111/j.1440-169x.2012.01357.x},
issn = {1440-169X},
year  = {2012},
date = {2012-01-01},
journal = {Development, Growth \& Differentiation},
volume = {54},
number = {5},
pages = {546--556},
abstract = {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 (6\textendash100%) 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Ochiai:2012gy,
title = {Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos},
author = {Hiroshi Ochiai and Naoaki Sakamoto and Kazumasa Fujita and Masatoshi Nishikawa and Ken-ichi Suzuki and Shinya Matsuura and Tatsuo Miyamoto and Tetsushi Sakuma and Tatsuo Shibata and Takashi Yamamoto},
url = {http://www.pnas.org/cgi/doi/10.1073/pnas.1202768109},
doi = {10.1073/pnas.1202768109},
issn = {0027-8424},
year  = {2012},
date = {2012-01-01},
journal = {Proceedings of the National Academy of Sciences},
volume = {109},
number = {27},
pages = {10915--10920},
abstract = {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 ∼1-kb homology arms and a 2A-histone H2B\textendashGFP 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Sakuma:2011hl,
title = {HpSumf1 is involved in the activation of sulfatases responsible for regulation of skeletogenesis during sea urchin development},
author = {Tetsushi Sakuma and Kazuya Ohnishi and Kazumasa Fujita and Hiroshi Ochiai and Naoaki Sakamoto and Takashi Yamamoto},
url = {http://link.springer.com/10.1007/s00427-011-0368-2},
doi = {10.1007/s00427-011-0368-2},
issn = {0949-944X},
year  = {2011},
date = {2011-01-01},
journal = {Development Genes and Evolution},
volume = {221},
number = {3},
pages = {157--166},
abstract = {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, Cα-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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Okamitsu:2010fs,
title = {Dicer is Required for the Normal Development of Sea Urchin, Hemicentrotus pulcherrimus},
author = {Yuka Okamitsu and Takashi Yamamoto and Takayoshi Fujii and Hiroshi Ochiai and Naoaki Sakamoto},
url = {http://www.bioone.org/doi/abs/10.2108/zsj.27.477},
doi = {10.2108/zsj.27.477},
issn = {0289-0003},
year  = {2010},
date = {2010-01-01},
journal = {Zoological Science},
volume = {27},
number = {6},
pages = {477--486},
abstract = {MicroRNAs are single-stranded RNA molecules with a length of 1925 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Ochiai:2010gn,
title = {Targeted mutagenesis in the sea urchin embryo using zinc‐finger nucleases},
author = {Hiroshi Ochiai and Kazumasa Fujita and Ken‐ichi Suzuki and Masatoshi Nishikawa and Tatsuo Shibata and Naoaki Sakamoto and Takashi Yamamoto},
url = {http://doi.wiley.com/10.1111/j.1365-2443.2010.01425.x},
doi = {10.1111/j.1365-2443.2010.01425.x},
issn = {1365-2443},
year  = {2010},
date = {2010-01-01},
journal = {Genes to Cells},
volume = {15},
number = {8},
pages = {875--885},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Fujii:2009ea,
title = {Role of the nanos homolog during sea urchin development},
author = {Takayoshi Fujii and Naoaki Sakamoto and Hiroshi Ochiai and Kazumasa Fujita and Yuka Okamitsu and Noriko Sumiyoshi and Takuya Minokawa and Takashi Yamamoto},
url = {http://doi.wiley.com/10.1002/dvdy.22074},
doi = {10.1002/dvdy.22074},
issn = {1097-0177},
year  = {2009},
date = {2009-01-01},
journal = {Developmental Dynamics},
volume = {238},
number = {10},
pages = {2511--2521},
abstract = {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\textendash2521, 2009. © 2009 Wiley-Liss, Inc.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Karasawa:2009gt,
title = {Suppressor of Hairless (Su(H)) is Required for Foregut Development in the Sea Urchin Embryo},
author = {Koji Karasawa and Naoaki Sakamoto and Kazumasa Fujita and Hiroshi Ochiai and Takayoshi Fujii and Koji Akasaka and Takashi Yamamoto},
url = {http://www.bioone.org/doi/abs/10.2108/zsj.26.686},
doi = {10.2108/zsj.26.686},
issn = {0289-0003},
year  = {2009},
date = {2009-01-01},
journal = {Zoological Science},
volume = {26},
number = {10},
pages = {686--690},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Ochiai:2008do,
title = {Analysis of cis-regulatory elements controlling spatio-temporal expression of T-brain gene in sea urchin, Hemicentrotus pulcherrimus},
author = {Hiroshi Ochiai and Naoaki Sakamoto and Asuka Momiyama and Koji Akasaka and Takashi Yamamoto},
url = {http://linkinghub.elsevier.com/retrieve/pii/S092547730700189X},
doi = {10.1016/j.mod.2007.10.009},
issn = {0925-4773},
year  = {2008},
date = {2008-01-01},
journal = {Mechanisms of Development},
volume = {125},
number = {1-2},
pages = {2--17},
abstract = {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 ∼7kb 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}

@article{Ochiai:2008ix,
title = {The Ars insulator facilitates I‐SceI meganuclease‐mediated transgenesis in the sea urchin embryo},
author = {Hiroshi Ochiai and Naoaki Sakamoto and Kenichi Suzuki and Koji Akasaka and Takashi Yamamoto},
url = {http://doi.wiley.com/10.1002/dvdy.21690},
doi = {10.1002/dvdy.21690},
issn = {1097-0177},
year  = {2008},
date = {2008-01-01},
journal = {Developmental Dynamics},
volume = {237},
number = {9},
pages = {2475--2482},
abstract = {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\textendashspecific 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\textendashmediated 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\textendashmediated method using the Ars insulator is advantageous for transgenesis in the sea urchin embryo. Developmental Dynamics 237:2475\textendash2482, 2008. © 2008 Wiley-Liss, Inc.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}