| 1. |
STREAMING-tag system reveals spatiotemporal relationships between transcriptional regulatory factors and transcriptional activity. |
| 2. |
Haruka Matsumori, Kenji Watanabe, Hiroaki Tachiwana, Tomoko Fujita, Yuma Ito, Makio Tokunaga, Kumiko Sakata-Sogawa, Hiroko Osakada, Tokuko Haraguchi, Akinori Awazu, Hiroshi Ochiai, Yuka Sakata, Koji Ochiai, Tsutomu Toki, Etsuro Ito, Ilya G Goldberg, Kazuaki Tokunaga, Mitsuyoshi Nakao, Noriko Saitoh, Ribosomal protein L5 facilitates rDNA-bundled condensate and nucleolar assembly, Life Science Alliance, 10.26508/lsa.202101045, 5, 7, e202101045, 2022.03. |
| 3. |
Silvia Natsuko Akutsu, Tatsuo Miyamoto, Daiju Oba, Keita Tomioka, Hiroshi Ochiai, Hirofumi Ohashi, Shinya Matsuura, iPSC reprogramming-mediated aneuploidy correction in autosomal trisomy syndromes, PLoS One, 10.1371/journal.pone.0264965, 17, 3, e0264965, 2022.03. |
| 4. |
Hiroshi Ochiai, Tetsutaro Hayashi, Mana Umeda, Mika Yoshimura, Akihito Harada, Yukiko Shimizu, Kenta Nakano, Noriko Saitoh, Zhe Liu, Takashi Yamamoto, Tadashi Okamura, Yasuyuki Ohkawa, Hiroshi Kimura, Itoshi Nikaido, Genome-wide kinetic properties of transcriptional bursting in mouse embryonic stem cells., Science Advances, 10.1126/sciadv.aaz6699, 6, 25, eaaz6699, 2020.06, 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.. |
| 5. |
Tatsuo Miyamoto, Kosuke Hosoba, Takeshi Itabashi, Atsuko H Iwane, Silvia Natsuko Akutsu, Hiroshi Ochiai, Yumiko Saito, Takashi Yamamoto, Shinya Matsuura, Insufficiency of ciliary cholesterol in hereditary Zellweger syndrome, The EMBO Journal, 10.15252/embj.2019103499, e103499, 2020.05. |
| 6. |
Soya Shinkai, Masaki Nakagawa, Takeshi Sugawara, Yuichi Togashi, Hiroshi Ochiai, Ryuichiro Nakato, Yuichi Taniguchi, Shuichi Onami, PHi-C: deciphering Hi-C data into polymer dynamics, NAR Genomics and Bioinformatics, 10.1093/nargab/lqaa020, 2, 2, lqaa020, 2020.03. |
| 7. |
Sungrim Seirin-Lee, Fumitaka Osakada, Junichi Takeda, Satoshi Tashiro, Ryo Kobayashi, Takashi Yamamoto, Hiroshi Ochiai, Role of dynamic nuclear deformation on genomic architecture reorganization, PLOS Computational Biology, 10.1371/journal.pcbi.1007289, 15, 9, e1007289-e1007289, 2019.09. |
| 8. |
Jieru Li, Ankun Dong, Kamola Saydaminova, Hill Chang, Guanshi Wang, Hiroshi Ochiai, Takashi Yamamoto, Alexandros Pertsinidis, Single-Molecule Nanoscopy Elucidates RNA Polymerase II Transcription at Single Genes in Live Cells, Cell, 10.1016/j.cell.2019.05.029, 178, 491-506, 2019.05. |
| 9. |
Masaya Matsushita, Hiroshi Ochiai, Ken-ichi T. Suzuki, Sayaka Hayashi, Takashi Yamamoto, Akinori Awazu, Naoaki Sakamoto, Dynamic changes in the interchromosomal interaction of early histone gene loci during development of sea urchin, JOURNAL OF CELL SCIENCE, 10.1242/jcs.206862, 130, 24, 4097-4107, 2017.12, 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.. |
| 10. |
Satoru Ishihara, Naoe Kotomura, Naoki Yamamoto, Hiroshi Ochiai, Ligation-mediated PCR with a back-to-back adapter reduces amplification bias resulting from variations in GC content, ANALYTICAL BIOCHEMISTRY, 10.1016/j.ab.2017.05.011, 531, 15, 37-44, 2017.08, 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.. |
| 11. |
Hiroshi Ochiai, Takeshi Sugawara, Takashi Yamamoto, Simultaneous live imaging of the transcription and nuclear position of specific genes, NUCLEIC ACIDS RESEARCH, 10.1093/nar/gkv624, 43, 19, e127, 2015.10, 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.. |
| 12. |
Tatsuo Miyamoto, Kosuke Hosoba, Hiroshi Ochiai, Ekaterina Royba, Hideki Izumi, Tetsushi Sakuma, Takashi Yamamoto, Brian David Dynlacht, Shinya Matsuura, The Microtubule-Depolymerizing Activity of a Mitotic Kinesin Protein KIF2A Drives Primary Cilia Disassembly Coupled with Cell Proliferation, CELL REPORTS, 10.1016/j.celrep.2015.01.003, 10, 5, 664-673, 2015.02, 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.. |
| 13. |
Hiroshi Ochiai, Takeshi Sugawara, Tetsushi Sakuma, Takashi Yamamoto, Stochastic promoter activation affects Nanog expression variability in mouse embryonic stem cells, SCIENTIFIC REPORTS, 10.1038/srep07125, 4, 7125, 2014.11, 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.. |
| 14. |
Hiroshi Ochiai, Tatsuo Miyamoto, Akinori Kanai, Kosuke Hosoba, Tetsushi Sakuma, Yoshiki Kudo, Keiko Asami, Atsushi Ogawa, Akihiro Watanabe, Tadashi Kajii, Takashi Yamamoto, Shinya Matsuura, TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUB1B as causative of PCS ( MVA) syndrome, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.1317008111, 111, 4, 1461-1466, 2014.01, 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.. |
| 15. |
Tetsushi Sakuma, Hiroshi Ochiai, Takehito Kaneko, Tomoji Mashimo, Daisuke Tokumasu, Yuto Sakane, Ken-Ichi Suzuki, Tatsuo Miyamoto, Naoaki Sakamoto, Shinya Matsuura, Takashi Yamamoto, Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity, Scientific Reports, 10.1038/srep03379, 3, 3379, 2013.11, 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.. |
| 16. |
Ken-ichi T. Suzuki, Yukiko Isoyama, Keiko Kashiwagi, Tetsushi Sakuma, Hiroshi Ochiai, Naoaki Sakamoto, Nobuaki Furuno, Akihiko Kashiwagi, Takashi Yamamoto, High efficiency TALENs enable F0 functional analysis by targeted gene disruption in Xenopus laevis embryos, BIOLOGY OPEN, 10.1242/bio.20133855, 2, 5, 448-452, 2013.05, 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.. |
| 17. |
Tetsushi Sakuma, Sayaka Hosoi, Knut Woltjen, Ken-Ichi Suzuki, Keiko Kashiwagi, Housei Wada, Hiroshi Ochiai, Tatsuo Miyamoto, Narudo Kawai, Yasunori Sasakura, Shinya Matsuura, Yasushi Okada, Atsuo Kawahara, Shigeo Hayashi, Takashi Yamamoto, Efficient TALEN construction and evaluation methods for human cell and animal applications, Genes to Cells, 10.1111/gtc.12037, 18, 4, 315-326, 2013.04, 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.. |
| 18. |
Xiaohong Song, Yuya Sato, Athary Felemban, Ayako Ito, Mahmud Hossain, Hiroshi Ochiai, Takashi Yamamoto, Kiyotoshi Sekiguchi, Hideaki Tanaka, Kunimasa Ohta, Equarin is involved as an FGF signaling modulator in chick lens differentiation, DEVELOPMENTAL BIOLOGY, 10.1016/j.ydbio.2012.05.029, 368, 1, 109-117, 2012.08, 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.. |
| 19. |
Takahito Watanabe, Hiroshi Ochiai, Tetsushi Sakuma, Hadley W. Horch, Naoya Hamaguchi, Taro Nakamura, Tetsuya Bando, Hideyo Ohuchi, Takashi Yamamoto, Sumihare Noji, Taro Mito, Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases, NATURE COMMUNICATIONS, 10.1038/ncomms2020, 3, 1017, 2012.08, 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.. |
| 20. |
Hiroshi Ochiai, Naoaki Sakamoto, Kazumasa Fujita, Masatoshi Nishikawa, Ken-ichi Suzuki, Shinya Matsuura, Tatsuo Miyamoto, Tetsushi Sakuma, Tatsuo Shibata, Takashi Yamamoto, Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 10.1073/pnas.1202768109, 109, 27, 10915-10920, 2012.07, 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.. |
| 21. |
Narudo Kawai, Hiroshi Ochiai, Tetsushi Sakuma, Lixy Yamada, Hitoshi Sawada, Takashi Yamamoto, Yasunori Sasakura, Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases, DEVELOPMENT GROWTH & DIFFERENTIATION, 10.1111/j.1440-169X.2012.01355.x, 54, 5, 535-545, 2012.06, 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.. |
| 22. |
Satoshi Ansai, Hiroshi Ochiai, Yuta Kanie, Yasuhiro Kamei, Yuki Gou, Takeshi Kitano, Takashi Yamamoto, Masato Kinoshita, Targeted disruption of exogenous EGFP gene in medaka using zinc-finger nucleases, DEVELOPMENT GROWTH & DIFFERENTIATION, 10.1111/j.1440-169X.2012.01357.x, 54, 5, 546-556, 2012.06, 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.. |
| 23. |
Tetsushi Sakuma, Kazuya Ohnishi, Kazumasa Fujita, Hiroshi Ochiai, Naoaki Sakamoto, Takashi Yamamoto, HpSumf1 is involved in the activation of sulfatases responsible for regulation of skeletogenesis during sea urchin development, DEVELOPMENT GENES AND EVOLUTION, 10.1007/s00427-011-0368-2, 221, 3, 157-166, 2011.08, 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.. |
| 24. |
Hiroshi Ochiai, Kazumasa Fujita, Ken-ichi Suzuki, Masatoshi Nishikawa, Tatsuo Shibata, Naoaki Sakamoto, Takashi Yamamoto, Targeted mutagenesis in the sea urchin embryo using zinc-finger nucleases, GENES TO CELLS, 10.1111/j.1365-2443.2010.01425.x, 15, 8, 875-885, 2010.08, 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.. |
| 25. |
Yuka Okamitsu, Takashi Yamamoto, Takayoshi Fujii, Hiroshi Ochiai, Naoaki Sakamoto, Dicer is Required for the Normal Development of Sea Urchin, Hemicentrotus pulcherrimus, ZOOLOGICAL SCIENCE, 10.2108/zsj.27.477, 27, 6, 477-486, 2010.06, 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.. |
| 26. |
Takayoshi Fujii, Naoaki Sakamoto, Hiroshi Ochiai, Kazumasa Fujita, Yuka Okamitsu, Noriko Sumiyoshi, Takuya Minokawa, Takashi Yamamoto, Role of the Nanos Homolog During Sea Urchin Development, DEVELOPMENTAL DYNAMICS, 10.1002/dvdy.22074, 238, 10, 2511-2521, 2009.10, 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.. |
| 27. |
Koji Karasawa, Naoaki Sakamoto, Kazumasa Fujita, Hiroshi Ochiai, Takayoshi Fujii, Koji Akasaka, Takashi Yamamoto, Suppressor of Hairless (Su(H)) is Required for Foregut Development in the Sea Urchin Embryo, ZOOLOGICAL SCIENCE, 10.2108/zsj.26.686, 26, 10, 686-690, 2009.10, 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.. |
| 28. |
Hiroshi Ochiai, Naoaki Sakamoto, Kenichi Suzuki, Koji Akasaka, Takashi Yamamoto, The Ars insulator facilitates I-SceI meganuclease-mediated transgenesis in the sea urchin embryo, DEVELOPMENTAL DYNAMICS, 10.1002/dvdy.21690, 237, 9, 2475-2482, 2008.09, 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.. |
| 29. |
Hiroshi Ochiai, Naoaki Sakamoto, Asuka Momiyama, Koji Akasaka, Takashi Yamamoto, Analysis of cis-regulatory elements controlling spatio-temporal expression of T-brain gene in sea urchin, Hemicentrotus pulcherrimus, MECHANISMS OF DEVELOPMENT, 10.1016/j.mod.2007.10.009, 125, 1-2, 2-17, 2008.01, 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.. |
