Tatsuya Takemoto : エレクトロポレーション法による簡便なゲノム編集マウス作製法, Mar. 2016.
2.
Tatsuya Takemoto : Regulation of Axial Stem Cells Deriving Neural and Mesodermal Tissues During Posterior Axial Elongation, Springer, Mar. 2014.
Academic Paper (Judged Full Paper):
1.
Yudai Hatakeyama, Nen Saito, Yusuke Mii, Ritsuko Takada, Takuma Shinozuka, Tatsuya Takemoto, Honda Naoki and Shinji Takada : Intercellular exchange of Wnt ligands reduces cell population heterogeneity during embryogenesis, Nature Communications, Vol.14, No.1, 1-16, 2023.
(Summary)
Wnt signaling is required to maintain bipotent progenitors for neural and paraxial mesoderm cells, the neuromesodermal progenitor (NMP) cells that reside in the epiblast and tailbud. Since epiblast/tailbud cells receive Wnt ligands produced by one another, this exchange may average out the heterogeneity of Wnt signaling levels among these cells. Here, we examined this possibility by replacing endogenous Wnt3a with a receptor-fused form that activates signaling in producing cells, but not in neighboring cells. Mutant mouse embryos show a unique phenotype in which maintenance of many NMP cells is impaired, although some cells persist for long periods. The epiblast cell population of these embryos increases heterogeneity in Wnt signaling levels as embryogenesis progresses and are sensitive to retinoic acid, an endogenous antagonist of NMP maintenance. Thus, mutual intercellular exchange of Wnt ligands in the epiblast cell population reduces heterogeneity and achieves robustness to environmental stress.
Takumi Maruhashi, Daisuke Sugiura, Il-mi Okazaki, Kenji Shimizu, K Takeo Maeda, Jun Ikubo, Harunori Yoshikawa, Katsumi Maenaka, Naozumi Ishimaru, Hidetaka Kosako, Tatsuya Takemoto and Taku Okazaki : Binding of LAG-3 to stable peptide-MHC class II limits T cell function and suppresses autoimmunity and anti-cancer immunity., Immunity, Vol.55, No.5, 912-924.e8, 2022.
(Summary)
Lymphocyte activation gene-3 (LAG-3) is a potent inhibitory co-receptor; yet, its functional ligand remains elusive, with distinct potential ligands identified. Here, we investigated the relative contribution of potential ligands, stable peptide-MHC class II complexes (pMHCII) and fibrinogen-like protein 1 (FGL1), to LAG-3 activity invitro and invivo. Binding of LAG-3 to stable pMHCII but not to FGL1 induced Tcell suppression invitro. Consistently, LAG-3 mutants lacking FGL1-binding capacity but not those lacking stable pMHCII-binding capacity retained suppressive activity invitro. Accordingly, targeted disruption of stable pMHCII- but not FGL1-binding capacity of LAG-3 in NOD mice recapitulated diabetes exacerbation by LAG-3 deficiency. Additionally, the loss of stable pMHCII-binding capacity of LAG-3 augmented anti-cancer immunity comparably with LAG-3 deficiency in C57BL/6 mice. These results identify stable pMHCII as a functional ligand of LAG-3 both in autoimmunity and anti-cancer immunity. Thus, stable pMHCII-LAG-3 interaction is a potential therapeutic target in human diseases.
(Keyword)
Animals / Antigens, CD / Autoimmunity / Histocompatibility Antigens Class II / Ligands / Mice / Mice, Inbred C57BL / Mice, Inbred NOD / Neoplasms / Peptides / T-Lymphocytes
Shimizu Kenji, Daisuke Sugiura, Il-mi Okazaki, Takumi Maruhashi, Tatsuya Takemoto and Taku Okazaki : PD-1 preferentially inhibits the activation of low-affinity T cells, Proceedings of the National Academy of Sciences of the United States of America, Vol.118, No.35, e2107141118, 2021.
(Summary)
Anti-PD-1 therapies can activate tumor-specific T cells to destroy tumors. However, whether and how T cells with different antigen specificity and affinity are differentially regulated by PD-1 remain vaguely understood. Upon antigen stimulation, a variety of genes is induced in T cells. Recently, we found that T cell receptor (TCR) signal strength required for the induction of genes varies across different genes and PD-1 preferentially inhibits the induction of genes that require stronger TCR signal. As each T cell has its own response characteristics, inducibility of genes likely differs across different T cells. Accordingly, the inhibitory effects of PD-1 are also expected to differ across different T cells. In the current study, we investigated whether and how factors that modulate T cell responsiveness to antigenic stimuli influence PD-1 function. By analyzing TCRs with different affinities to peptide-MHC complexes (pMHC) and pMHCs with different affinities to TCR, we demonstrated that PD-1 inhibits the expression of TCR-inducible genes efficiently when TCR:pMHC affinity is low. In contrast, affinities of peptides to MHC and MHC expression levels did not affect PD-1 sensitivity of TCR-inducible genes although they markedly altered the dose responsiveness of T cells by changing the efficiency of pMHC formation, suggesting that the strength of individual TCR signal is the key determinant of PD-1 sensitivity. Accordingly, we observed a preferential expansion of T cells with low-affinity to tumor-antigen in PD-1-deficient mice upon inoculation of tumor cells. These results demonstrate that PD-1 imposes qualitative control of T cell responses by preferentially suppressing low-affinity T cells.
Shinichi Hayashi, Hitomi Suzuki and Tatsuya Takemoto : The nephric mesenchyme lineage of intermediate mesoderm is derived from Tbx6-expressing derivatives of neuro-mesodermal progenitors via BMP-dependent Osr1 function, Developmental Biology, Vol.478, 155-162, 2021.
(Summary)
In vertebrate embryos, the kidney primordium metanephros is formed from two distinct cell lineages, Wolffian duct and metanephric mesenchyme, which were classically grouped as intermediate mesoderm. Whereas the reciprocal interactions between these two cell populations in kidney development have been studied extensively, the mechanisms generating them remain elusive. Here, we show that the mouse cell lineage that forms nephric mesenchyme develops as a subpopulation of Tbx6-expressing mesodermal precursor derivatives of neuro-mesodermal progenitors (NMPs) under the condition of bone morphogenetic protein (BMP)-signal-dependent Osr1 expression. The Osr1-expressing nephric mesenchyme precursors were confirmed as descendants of NMPs because they were labeled by Sox2 N1 enhancer-EGFP. In Tbx6 mutant embryos, nephric mesenchyme changed its fate into neural tissues, which reflected its NMP origin. In Osr1 mutant embryos, the specific region of the Tbx6-expressing mesoderm precursor, which normally expresses Osr1 and develops into the nephric mesenchyme, instead expressed the somite marker FoxC2. BMP signaling activated Osr1 expression in a region of TBX6-expressing mesoderm and elicited nephric mesenchyme development. This study suggested a new model of cell lineage segregation during gastrulation.
Taichi Nakatani, Mitsuhiro Iwasaki, Atsuhiro Yamamichi, Yuta Yoshioka, Toshihiro Uesaka, Yuko Bitoh, Kosaku Maeda, Takumi Fukumoto, Tatsuya Takemoto and Hideki Enomoto : Point mutagenesis in mouse reveals contrasting pathogenetic effects between MEN2B- and Hirschsprung disease-associated missense mutations of the RET gene, Development Growth & Differentiation, Vol.4, No.62, 214-222, 2020.
(Summary)
Missense mutations of the RET gene have been identified in both multiple endocrine neoplasia (MEN) type 2A/B and Hirschsprung disease (HSCR: congenital absence of the enteric nervous system, ENS). Current consensus holds that MEN2A/B and HSCR are caused by activating and inactivating RET mutations, respectively. However, the biological significance of RET missense mutations in vivo has not been fully elucidated. In the present study, we introduced one MEN2B-associated (M918T) and two HSCR-associated (N394K and Y791F) RET missense mutations into the corresponding regions of the mouse Ret gene by genome editing (Ret , Ret and Ret ) and performed histological examinations of Ret-expressing tissues to understand the pathogenetic impact of each mutant in vivo. Ret mice displayed MEN2B-related phenotypes, including C-cell hyperplasia and abnormal enlargement of the primary sympathetic ganglia. Similar sympathetic phenotype was observed in Ret mice, demonstrating a strong pathogenetic effect of the Ret M918T by a single-allele expression. In contrast, no abnormality was found in the ENS of mice harboring the Ret N394K or Y791F mutation. Most surprisingly, single-allele expression of RET N394K or Y791F was sufficient for normal ENS development, indicating that these RET mutants exert largely physiological function in vivo. This study reveals contrasting pathogenetic effects between MEN2B- and HSCR-associated RET missense mutations, and suggests that some of HSCR-associated RET missense mutations are by themselves neither inactivating nor pathogenetic and require involvement of other gene mutations for disease expressivity.
Shiori Yamamoto, Yuji Uchida, Tomomi Ohtani, Erina Nozaki, Chunyang Yin, Yoshihiro Gotoh, Nayuta YakushijiKaminatsui, Tetsuya Higashiyama, Takamasa Suzuki, Tatsuya Takemoto, Shiraishi Yo-Ichi and Kuroiwa Atsushi : Hoxa13 regulates expression of common Hox target genes involved in cartilage development to coordinate the expansion of the autopodal anlage., Development Growth & Differentiation, Vol.61, No.3, 228-251, 2020.
(Summary)
To elucidate the role of Hox genes in limb cartilage development, we identified the target genes of HOXA11 and HOXA13 by ChIP-Seq. The ChIP DNA fragment contained evolutionarily conserved sequences and multiple highly conserved HOX binding sites. A substantial portion of the HOXA11 ChIP fragment overlapped with the HOXA13 ChIP fragment indicating that both factors share common targets. Deletion of the target regions neighboring Bmp2 or Tshz2 reduced their expression in the autopod suggesting that they function as the limb bud-specific enhancers. We identified the Hox downstream genes as exhibiting expression changes in the Hoxa13 knock out (KO) and Hoxd11-13 deletion double mutant (Hox13 dKO) autopod by Genechip analysis. The Hox downstream genes neighboring the ChIP fragment were defined as the direct targets of Hox. We analyzed the spatial expression pattern of the Hox target genes that encode two different categories of transcription factors during autopod development and Hox13dKO limb bud. (a) Bcl11a, encoding a repressor of cartilage differentiation, was expressed in the E11.5 autopod and was substantially reduced in the Hox13dKO. (b) The transcription factors Aff3, Bnc2, Nfib and Runx1t1 were expressed in the zeugopodal cartilage but not in the autopod due to the repressive or relatively weak transcriptional activity of Hox13 at E11.5. Interestingly, the expression of these genes was later observed in the autopodal cartilage at E12.5. These results indicate that Hox13 transiently suspends the cartilage differentiation in the autopodal anlage via multiple pathways until establishing the paddle-shaped structure required to generate five digits.
Machiko Teramoto, Ryo Sugawara, Katsura Minegishi, Masanori Uchikawa, Tatsuya Takemoto, Atsushi Kuroiwa, Yasuo Ishii and Hisato Kondoh : The absence of SOX2 in the anterior foregut alters the esophagus into trachea and bronchi in both epithelial and mesenchymal components., Biology Open, Vol.9, No.2, 2020.
Hideaki Iida, Yoko Furukawa, Machiko; Teramoto, Hitomi Suzuki, Tatsuya Takemoto, Masanori Uchikawa and Hisato Kondoh : Sox2 gene regulation via the D1 enhancer in embryonic neural tube and neural crest by the combined action of SOX2 and ZIC2., Genes to Cells, 2020.
(Summary)
The transcription factor (TF) SOX2 regulates various stem cells and tissue progenitors via functional interactions with cell type-specific partner TFs that co-bind to enhancer sequences. Neural progenitors are the major embryonic tissues where SOX2 assumes central regulatory roles. In order to characterize the partner TFs of SOX2 in neural progenitors, we investigated the regulation of the D1 enhancer of the Sox2 gene, which is activated in the embryonic neural tube (NT) and neural crest (NC), using chicken embryo electroporation. We identified essential TF binding sites for a SOX, and two ZIC TFs in the activation of the D1 enhancer. By comparison of dorso-ventral and antero-posterior patterns of D1 enhancer activation, and the effect of mutations on the enhancer activation patterns with TF expression patterns, we determined SOX2 and ZIC2 as the major D1 enhancer-activating TFs. Binding of these TFs to the D1 enhancer sequence was confirmed by chromatin immunoprecipitation analysis. The combination of SOX2 and ZIC2 TFs activated the enhancer in both the NT and NC. These results indicate that SOX2 and ZIC2, which have been known to play major regulatory roles in neural progenitors, do functionally cooperate. In addition, the recently demonstrated SOX2 expression during the NC development is accounted for at least partly by the D1 enhancer activity. Deletion of the D1 enhancer sequence from the mouse genome, however, did not affect the mouse development, indicating functional redundancies of other enhancers.
Tatsuya Takemoto : Zygote Electroporation for CRISPR/Cas9 Delivery to Generate Genetically Modified Mice, Methods in Molecular Biology, Vol.2050, 121-126, 2020.
(Summary)
The CRISPR/Cas9 system is a powerful tool for generation of genetically modified mice. In conventional protocols, Cas9 protein (or mRNA) and sgRNA are introduced into zygotes by microinjection. However, microinjection requires special skill and is too time-consuming to treat zygotes on a large scale. Recently, we have developed a simple electroporation method which generates genetically modified mice with high efficiency. Here, we describe our method GEEP (genome editing by electroporation of Cas9 protein). This method facilitates high-throughput genetic analysis of the mouse. This chapter describes the GEEP method to generate genetically modified mice.
(Keyword)
Animals / Animals, Genetically Modified / CRISPR-Associated Protein 9 / CRISPR-Cas Systems / Electroporation / Female / Male / Mice / RNA, Guide / Zygote
Daisuke Sugiura, Takumi Maruhashi, Il-mi Okazaki, Kenji Shimizu, Takeo K. Maeda, Tatsuya Takemoto and Taku Okazaki : Restriction of PD-1 function by -PD-L1/CD80 interactions is required for optimal T cell responses., Science, Vol.364, No.6440, 558-566, 2019.
(Summary)
Targeted blockade of PD-1 with immune checkpoint inhibitors can activate T cells to destroy tumors. PD-1 is believed to function mainly at the effector, but not in the activation, phase of T cell responses, yet how PD-1 function is restricted at the activation stage is currently unknown. Here we demonstrate that CD80 interacts with PD-L1 in cis on antigen-presenting cells (APCs) to disrupt PD-L1/PD-1 binding. Subsequently, PD-L1 cannot engage PD-1 to inhibit T cell activation when APCs express substantial amounts of CD80. In knock-in mice in which -PD-L1/CD80 interactions do not occur, tumor immunity and autoimmune responses were greatly attenuated by PD-1. These findings indicate that CD80 on APCs limits the PD-1 coinhibitory signal, while promoting CD28-mediated costimulation, and highlight critical components for induction of optimal immune responses.
Fuminori Tanihara, Maki Hirata, Nhien T. Nguyen, Quynh A. Le, Takayuki Hirano, Tatsuya Takemoto, Michiko Nakai, Dai-Ichiro Fuchimoto and Takeshige Otoi : Generation of PDX-1 mutant porcine blastocysts by introducing CRISPR/Cas9-system into porcine zygotes via electroporation., Animal Science Journal, Vol.90, No.1, 55-61, 2018.
(Summary)
Recently, we established the GEEP ("gene editing by electroporation of Cas9 protein") method, in which the CRISPR/Cas9 system, consisting of a Cas9 protein and single guide RNA (sgRNA), is introduced into pig zygotes by electroporation and thus induces highly efficient targeted gene disruption. In this study, we examined the effects of sgRNA on the blastocyst formation of porcine embryos and evaluated their genome-editing efficiency. To produce an animal model for diabetes, we targeted PDX-1 (pancreas duodenum homeobox 1), a gene that is crucial for pancreas development during the fetal period and whose monoallelic disruption impairs insulin secretion. First, Cas9 protein with different sgRNAs that targeted distinct sites in the PDX-1 exon 1 was introduced into in vitro-fertilized zygotes by the GEEP method. Of the six sgRNAs tested, three sgRNAs (sgRNA1, 2, and 3) successfully modified PDX-1 gene. The blastocyst formation rate of zygotes edited with sgRNA3 was significantly (p < 0.05) lower than that of control zygotes without the electroporation treatment. Our study indicates that the GEEP method can be successfully used to generate PDX-1 mutant blastocysts, but the development and the efficiency of editing the genome of zygotes may be affected by the sgRNA used for CRISPR/Cas9 system.
Fuminori Tanihara, Maki Hirata, Nhien Thi Nguyen, Quynh Anh Le, Takayuki Hirano, Tatsuya Takemoto, Michiko Nakai, Dai-Ichiro Fuchimoto and Takeshige Otoi : Generation of a TP53-modified porcine cancer model by CRISPR/Cas9-mediated gene modification in porcine zygotes via electroporation., PLoS ONE, Vol.13, No.10, e0206360, 2018.
(Summary)
TP53 (which encodes p53) is one of the most frequently mutated genes in cancers. In this study, we generated TP53-mutant pigs by gene editing via electroporation of the Cas9 protein (GEEP), a process that involves introducing the Cas9 protein and single-guide RNA (sgRNA) targeting exon 3 and intron 4 of TP53 into in vitro-fertilized zygotes. Zygotes modified by the sgRNAs were transferred to recipients, two of which gave birth to a total of 11 piglets. Of those 11 piglets, 9 survived. Molecular genetic analysis confirmed that 6 of 9 live piglets carried mutations in TP53, including 2 piglets with no wild-type (WT) sequences and 4 genetically mosaic piglets with WT sequences. One mosaic piglet had 142 and 151 bp deletions caused by a combination of the two sgRNAs. These piglets were continually monitored for 16 months and three of the genome-edited pigs (50%) exhibited various tumor phenotypes that we presumed were caused by TP53 mutations. Two mutant pigs with no WT sequences developed mandibular osteosarcoma and nephroblastoma. The mosaic pig with a deletion between targeting sites of two sgRNAs exhibited malignant fibrous histiocytoma. Tumor phenotypes of TP53 mosaic mutant pigs have not been previously reported. Our results indicated that the mutations caused by gene editing successfully induced tumor phenotypes in both TP53 mosaic- and bi-allelic mutant pigs.
Regulation of gene expression requires selective incorporation of histone H3 variant H3.3 into chromatin. Histone H3.3 has several subsidiary variants but their functions are unclear. Here we characterize the function of histone H3.3 sub-variant, H3mm7, which is expressed in skeletal muscle satellite cells. H3mm7 knockout mice demonstrate an essential role of H3mm7 in skeletal muscle regeneration. Chromatin analysis reveals that H3mm7 facilitates transcription by forming an open chromatin structure around promoter regions including those of myogenic genes. The crystal structure of the nucleosome containing H3mm7 reveals that, unlike the S57 residue of other H3 proteins, the H3mm7-specific A57 residue cannot form a hydrogen bond with the R40 residue of the cognate H4 molecule. Consequently, the H3mm7 nucleosome is unstable in vitro and exhibited higher mobility in vivo compared with the H3.3 nucleosome. We conclude that the unstable H3mm7 nucleosome may be required for proper skeletal muscle differentiation.
Katsutoshi Nishio, Fuminori Tanihara, T-V Nguyen, Toshiki Kunihara, M Nii, Maki Hirata, Tatsuya Takemoto and Takeshige Otoi : Effects of voltage strength during electroporation on the development and quality of in vitro-produced porcine embryos., Reproduction in Domestic Animals = Zuchthygiene, Vol.53, No.2, 313-318, 2017.
(Summary)
This study was conducted to determine suitable conditions for an experimental method in which the CRISPR/Cas9 system is introduced into in vitro-produced porcine zygotes by electroporation. In the first experiment, when putative zygotes derived from in vitro fertilization (IVF) were electroporated by either unipolar or bipolar pulses, keeping the voltage, pulse duration and pulse number fixed at 30 V/mm, 1 msec and five repeats, respectively, the rate of blastocyst formation from zygotes electroporated by bipolar pulses decreased compared to zygotes electroporated by unipolar pulses. In the second experiment, the putative zygotes were electroporated by electroporation voltages ranging from 20 V/mm-40 V/mm with five 1-msec unipolar pulses. The rate of cleavage and blastocyst formation of zygotes electroporated at 40 V/mm was significantly lower (p < .05) than that of zygotes electroporated at less than 30 V/mm. Moreover, the apoptotic nuclei indices of blastocysts derived from zygotes electroporated by voltages greater than 30 V/mm significantly increased compared with those from zygotes electroporated by voltages less than 25 V/mm (p < .05). When zygotes were electroporated with Cas9 mRNA and single-guide RNA (sgRNA) targeting site in the FGF10 exon 3, the proportions of blastocysts with targeted genomic sequences were 7.7% (2/26) and 3.6% (1/28) in the embryos derived from zygotes electroporated at 25 V/mm and 30 V/mm, respectively. Our results indicate that electroporation at 25 V/mm may be an acceptable condition for introducing Cas9 mRNA and sgRNA into pig IVF zygotes under which the viability of the embryos is not significantly affected.
Katsutoshi Nishio, Mado Yamazaki, Masayasu Taniguchi, Kazuhiko Besshi, Fumio Morita, Toshiki Kunihara, Fuminori Tanihara, Tatsuya Takemoto and Takeshige Otoi : Sensitivity of the meiotic stage to hyperthermia during in vitro maturation of porcine oocytes., Acta Veterinaria Hungarica, Vol.65, No.1, 115-123, 2017.
(Summary)
The present study was conducted to clarify whether the meiotic stage of porcine oocytes has the highest sensitivity to hyperthermia during in vitro maturation by evaluating meiotic competence and DNA damage. Oocytes were exposed to 41 °C for 12 h at various intervals during 48 h of maturation culture. When the oocytes were exposed to 41 °C from 12 to 24 h of the maturation culture, the proportion of oocytes reaching metaphase II (MII) decreased as compared to the control oocytes cultured at 38.5 °C (P < 0.05). Moreover, the proportions of DNA fragmentation in all oocytes exposed to 41 °C in each culture period after 12 h from the start of maturation culture were significantly higher (P < 0.05) than for the control oocytes. When the meiotic stage of oocytes cultured at 38.5 °C between 12 and 24 h was examined, the majority of oocytes remained at the germinal vesicle (GV) stage at 12 h and approximately half of the oocytes reached metaphase I (MI) at 24 h. These results indicate that the meiotic stage of porcine oocytes having the highest sensitivity to hyperthermia during in vitro maturation is a transition period from the GV stage to the MI stage.
(Keyword)
Animals / DNA Damage / Hot Temperature / In Vitro Oocyte Maturation Techniques / Meiosis / Oocytes / Swine
Uddin Myn Muhammad, Izumi Ohigashi, Ryo Motosugi, Tomomi Nakayama, Mie Sakata, Jun Hamazaki, Yasumasa Nishito, Immanuel Rode, Keiji Tanaka, Tatsuya Takemoto, Shigeo Murata and Yousuke Takahama : Foxn1-5t transcriptional axis controls CD8+ T-cell production in the thymus., Nature Communications, Vol.8, 14419, 2017.
(Summary)
The thymus is an organ that produces functionally competent T cells that protect us from pathogens and malignancies. Foxn1 is a transcription factor that is essential for thymus organogenesis; however, the direct target for Foxn1 to actuate thymic T-cell production is unknown. Here we show that a Foxn1-binding cis-regulatory element promotes the transcription of β5t, which has an essential role in cortical thymic epithelial cells to induce positive selection of functionally competent CD8(+) T cells. A point mutation in this genome element results in a defect in β5t expression and CD8(+) T-cell production in mice. The results reveal a Foxn1-β5t transcriptional axis that governs CD8(+) T-cell production in the thymus.
Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs.
L Do, M Wittayarat, T Terazono, Y Sato, M Taniguchi, Fuminori Tanihara, Tatsuya Takemoto, Y Kazuki, K Kazuki, M Oshimura and Takeshige Otoi : Effects of duration of electric pulse on in vitro development of cloned cat embryos with human artificial chromosome vector., Reproduction in Domestic Animals = Zuchthygiene, Vol.51, No.6, 1039-1043, 2016.
N Kurniani Karja Wayan, M Fahrudin, MA Setiadi, LI Tumbelaka, R Sudarwati, YT Hastuti, BH Mulia, A Widianti, K Sultan, T Terazono, Z Namula, M Taniguchi, Fuminori Tanihara, Tatsuya Takemoto, K Kikuchi, Y Sato and Takeshige Otoi : Characteristics and fertility of sumatran tiger spermatozoa cryopreserved with different sugars., Cryo Letters, Vol.37, No.4, 264-271, 2016.
(Summary)
Cryopreservation of semen is one of the most important methods for the preservation of endangered tigers. This study evaluated the effects of sugar supplementation on the cryosurvival of spermatozoa from Sumatran tigers (Panthera tigris sumatrae). The post-thaw characteristics and fertility of spermatozoa cryopreserved with different sugars (glucose, lactose, and trehalose) were evaluated using heterologous in-vitro fertilisation with cat oocytes. All parameters of post-thaw spermatozoa significantly decreased as compared with those of fresh spermatozoa. The index of sperm motility for semen cryopreserved with lactose was significantly higher than that for semen cryopreserved with trehalose. The percentage of total fertilisation for tiger spermatozoa cryopreserved with trehalose was significantly lower than that for control cat spermatozoa. Our findings indicated that supplementation with lactose or glycerol as the main sugar in the egg yolk extender resulted in a better motility and fertility potential for post-thawed spermatozoa.
M Hashimoto, Y Yamashita and Tatsuya Takemoto : Electroporation of Cas9 protein/sgRNA into early pronuclear zygotes generates non-mosaic mutants in the mouse., Developmental Biology, Vol.418, No.1, 1-9, 2016.
(Summary)
The CRISPR/Cas9 system is a powerful tool for elucidating the roles of genes in a wide variety of organisms including mice. To obtain genetically modified embryos or mice by this method, Cas9 mRNA and sgRNA are usually introduced into zygotes by microinjection or electroporation. However, most mutants generated with this method are genetically mosaic, composed of several types of cells carrying different mutations, which complicates phenotype analysis in founder embryos or mice. To simplify the analysis and to elucidate the roles of genes involved in developmental processes, a method for producing non-mosaic mutants is needed. Here, we established a method for generating non-mosaic mouse mutant embryos. We introduced Cas9 protein and sgRNA into in vitro fertilized (IVF) zygotes by electroporation, which enabled the genome editing to occur before the first replication of the mouse genome. As a result, all of the cells in the mutant carried the same set of mutations. This method solves the problem of mosaicism/allele complexity in founder mutant embryos or mice generated by the CRIPSR/Cas9 system.
H Kondoh, S Takada and Tatsuya Takemoto : Axial level-dependent molecular and cellular mechanisms underlying the genesis of the embryonic neural plate., Development Growth & Differentiation, Vol.58, No.5, 427-436, 2016.
(Summary)
The transcription factor gene Sox2, centrally involved in neural primordial regulation, is activated by many enhancers. During the early stages of embryonic development, Sox2 is regulated by the enhancers N2 and N1 in the anterior neural plate (ANP) and posterior neural plate (PNP), respectively. This differential use of the enhancers reflects distinct regulatory mechanisms underlying the genesis of ANP and PNP. The ANP develops directly from the epiblast, triggered by nodal signal inhibition, and via the combined action of TFs SOX2, OTX2, POU3F1, and ZIC2, which promotes the the ANP development and inhibits other cell lineages. In contrast, the PNP is derived from neuromesodermal bipotential axial stem cells that develop into the neural plate when Sox2 is activated by the N1 enhancer, whereas they develop into the paraxial mesoderm when the N1 enhancer is repressed by the action of TBX6. The axial stem cells are maintained by the activity of WNT3a and T (Brachyury). However, at axial levels more anterior to the 8th somites (cervical levels), the development of both the neural plate and somite proceeds in the absence of WNT3a, T, or TBX6. These observations indicate that distinct molecular and cellular mechanisms determine neural plate genesis based on the axial level, and contradict the classical concept of the term "neural induction," which assumes a pan-neural plate mechanism.
Tatsuya Takemoto, Abe Takaya, Kiyonari Hiroshi, Nakao Kazuki, Furuta Yasuhide, Suzuki Hitomi, Takada Shinji, Fujimori Toshihiko and Kondoh Hisato : R26-WntVis reporter mice showing graded response to Wnt signal levels, Genes to Cells, 2016.
(Summary)
The canonical Wnt signaling pathway plays a major role in the regulation of embryogenesis and organogenesis, where signal strength-dependent cellular responses are of particular importance. To assess Wnt signal levels in individual cells, and to circumvent the integration site-dependent bias shown in previous Wnt reporter lines, we constructed a new Wnt signal reporter mouse line R26-WntVis. Heptameric TCF/LEF1 binding sequences were combined with a viral minimal promoter to confer a graded response to the reporter depending on Wnt signal strengths. The histone H2B-EGFP fusion protein was chosen as the fluorescent reporter to facilitate single-cell resolution analyses. This WntVis reporter gene was then inserted into the ROSA26 locus in an orientation opposite to that of the endogenous gene. The R26-WntVis allele was introduced into Wnt3a(-/-) and Wnt3a(vt/-) mutant mouse embryos and compared with wild-type embryos to assess its performance. The R26-WntVis reporter was activated in known Wnt-dependent tissues and responded in a graded fashion to signal intensity. This analysis also indicated that the major Wnt activity early in embryogenesis switched from Wnt3 to Wnt3a around E7.5. The R26-WntVis mouse line will be widely useful for the study of Wnt signal-dependent processes.
Morita Yasuhiro, Taniguchi Masayasu, Fuminori Tanihara, Ito Aya, Namula Zhao, DO Thi Kim Lanh, Takagi Mitsuhiro, Tatsuya Takemoto and Takeshige Otoi : The optimal period of Ca-EDTA treatment for parthenogenetic activation of porcine oocytes during maturation culture, The Journal of Veterinary Medical Science, 2016.
(Summary)
The changes triggered by sperm-induced activation of oocytes, which are required for normal oocyte development, can be mediated by other agents, thereby inducing the parthenogenesis. In this study, we exposed porcine oocytes to 1 mM Ca-EDTA, a metal-ion chelator, at various intervals during 48 hr of in vitro maturation to determine the optimum period of Ca-EDTA treatment for parthenogenetic activation. When the oocytes were cultured with or without Ca-EDTA from 36 hr (post-12), 24 hr (post-24), 12 hr (post-36) and 0 hr (post-48) after the start of maturation culture, the blastocyst formation rates were significantly higher (P<0.05) in the post-24, post-36 and post-48 groups (3.3%, 4.0%, and 2.6%, respectively) than those in the control group without treatment (0%). Furthermore, when the oocytes were cultured with Ca-EDTA for 0 hr (control), 12 hr (pre-12), 24 hr (pre-24), 36 hr (pre-36) and 48 hr (pre-48) from the start of maturation culture, the oocytes formed blastocysts only in the pre-36 and pre-48 groups (0.4% or 0.8%, respectively). Pronuclei (<66.7%) were observed only when the periods of Ca-EDTA treatment were more than 12 hr during maturation culture. In the control group, no pronuclei were detected. Our findings demonstrate that porcine immature oocytes can be parthenogenetically activated by Ca-EDTA treatment for at least 24 hr to 36 hr during maturation culture, leading to pronucleus formation followed by the formation of blastocysts.
Masakazu Hashimoto and Tatsuya Takemoto : Electroporation enables the efficient mRNA delivery into the mouse zygotes and facilitates CRISPR/Cas9-based genome editing, Scientific Reports, Vol.5, 11315, 2015.
(Summary)
Recent use of the CRISPR/Cas9 system has dramatically reduced the time required to produce mutant mice, but the involvement of a time-consuming microinjection step still hampers its application for high-throughput genetic analysis. Here we developed a simple, highly efficient, and large-scale genome editing method, in which the RNAs for the CRISPR/Cas9 system are electroporated into zygotes rather than microinjected. We used this method to perform single-stranded oligodeoxynucleotide (ssODN)-mediated knock-in in mouse embryos. This method facilitates large-scale genetic analysis in the mouse.
Megumi Yoshida, Masanori Uchikawa, Karine Rizzoti, Robin Lovell-Badge, Tatsuya Takemoto and Hisato Kondoh : Regulation of mesodermal precursor production by low-level expression of B1 Sox genes in the caudal lateral epiblast., Mechanisms of Development, Vol.132, 59-68, 2014.
(Summary)
High expression of the B1 Sox genes, Sox2 and Sox3, is associated with the development of definitive neural primordia, the neural plates, in early stage embryos. However, in the caudal lateral epiblast (CLE) where axial stem cells reside, Sox2 and Sox3 are expressed at low levels, together with Brachyury. Because axial stem cells are the bipotential precursors of the neural plate and paraxial mesoderm, we investigated the possibility that low-level B1 Sox expression in CLE may regulate the fate of axial stem cells. We combined the genetic conditions of Sox3-null and Sox2 N1 enhancer homozygous deletion (Sox2(N1/N1)) to decrease B1 Sox expression in CLE. At 5-7 somite stages of mouse embryogenesis, these genetic manipulations caused approximately 30% higher production of paraxial mesodermal precursors, resulting in the development of larger somites. Analysis of mitotic cell populations suggested that decrease of B1 Sox expression in CLE does not activate cell proliferation but promotes cell migration into the mesodermal compartment. Thus, the low-level B1 Sox expression in CLE regulates axial stem cells to adjust the production of paraxial mesoderm precursors to an appropriate level.
Tatsuya Takemoto : Mechanism of cell fate choice between neural and mesodermal development during early embryogenesis., Congenital Anomalies, Vol.53, No.2, 61-66, 2013.
(Summary)
During early embryogenesis, Sox2 expression distinguishes the neural plate from other embryonic domains, suggesting that the mechanism underlying the activation of the Sox2 gene is highly relevant to the development of this tissue. At the earliest stages of neural plate development, the Sox2 enhancer N1 regulates Sox2 expression in the extending posterior end of the neural plate. The N1 enhancer is initially activated in the axial stem cells, bipotential precursors of both neural and mesodermal lineages, therefore the activation does not immediately lead to Sox2 expression. A population of axial stem cells that remains in the superficial layer starts expressing Sox2, whereas another population that migrates through the primitive streak loses the N1 activity and becomes mesoderm. Multiple signaling cascades and transcription factors, including Wnt, fibroblast growth factor (FGF), bone morphogenetic protein (BMP) and Tbx6, are responsible for the regulation of Sox2 expression in axial stem cells to guide the development of the posterior neural plate and paraxial mesoderm.
Hisato Kondoh and Tatsuya Takemoto : Axial stem cells deriving both posterior neural and mesodermal tissues during gastrulation., Current Opinion in Genetics & Development, Vol.22, No.4, 374-380, 2012.
(Summary)
The posterior neural plate is primarily derived from the axial stem cells bipotential for neural and paraxial mesodermal development, which reside in the caudal lateral epiblast (CLE) of gastrulating amniote embryos. This process has been demonstrated only recently through cell lineage analyses and determination of Sox2 activation mechanisms. The alternative developmental pathways depend on the activation of either transcription factor genes Sox2 (neural) or Tbx6 (mesodermal); the latter occurs in association with cell ingression through the primitive streak. Tbx6 mutant embryos develop ectopic neural tubes at the expense of the paraxial mesoderm, as Sox2 is expressed even after cell ingression. While producing alternative somatic cell populations, the axial stem cells proliferatively maintain themselves through a process dependent on the Brachyury-Wnt3a coregulatory loop, and even contribute to a fraction of later stem cells of the tail bud in the chordoneural hinge (CNH). Experimental evidence for the above processes is discussed, and unsolved problems indicated.
Masanori Uchikawa, Megumi Yoshida, Makiko Iwafuchi-Doi, Kazunari Matsuda, Yoshiko Ishida, Tatsuya Takemoto and Hisato Kondoh : B1 and B2 Sox gene expression during neural plate development in chicken and mouse embryos: universal versus species-dependent features., Development Growth & Differentiation, Vol.53, No.6, 761-771, 2011.
(Summary)
Cumulative evidence now indicates pivotal roles for the group B1 Sox genes, Sox1, Sox2 and Sox3 in the genesis and development of neural primordia. Shared functions for the Sox1, Sox2 and Sox3 protein products have also been indicated. This emphasizes the importance and integral role of the group B1 Sox genes in regulating the neural primordia. We here review what is currently known about the expression patterns of both the group B1 Sox genes and the related group B2 Sox21 gene during the embryonic stages when the neural plate develops. These expression profiles are compared between the chicken and mouse embryos, both representatives of amniote species. This comparison indicates a gross conservation of the regulation of individual Sox genes, yet also demonstrates the existence of species-dependent variations, which should be taken into account when data from different species are being compared. To link the expression patterns and transcriptional regulation of these genes, contribution of gene-specific enhancers are discussed. The regulation of B1 Sox genes in the axial stem cells, the common precursors to the posterior neural plate and paraxial mesoderm and located at the posterior end of developing neural plate, is also highlighted in this review. This article thus provides a guide to performing readouts of B1/B2 Sox expression data during neural plate development in amniotes.
(Keyword)
Embryonic Development / Gene Expression Profiling / Gene Expression Regulation
Tatsuya Takemoto, Masanori Uchikawa, Megumi Yoshida, M Donald Bell, Robin Lovell-Badge, E Virginia Papaioannou and Hisato Kondoh : Tbx6-dependent Sox2 regulation determines neural or mesodermal fate in axial stem cells., Nature, Vol.470, No.7334, 394-398, 2011.
(Summary)
The classical view of neural plate development held that it arises from the ectoderm, after its separation from the mesodermal and endodermal lineages. However, recent cell-lineage-tracing experiments indicate that the caudal neural plate and paraxial mesoderm are generated from common bipotential axial stem cells originating from the caudal lateral epiblast. Tbx6 null mutant mouse embryos which produce ectopic neural tubes at the expense of paraxial mesoderm must provide a clue to the regulatory mechanism underlying this neural versus mesodermal fate choice. Here we demonstrate that Tbx6-dependent regulation of Sox2 determines the fate of axial stem cells. In wild-type embryos, enhancer N1 of the neural primordial gene Sox2 is activated in the caudal lateral epiblast, and the cells staying in the superficial layer sustain N1 activity and activate Sox2 expression in the neural plate. In contrast, the cells destined to become mesoderm activate Tbx6 and turn off enhancer N1 before migrating into the paraxial mesoderm compartment. In Tbx6 mutant embryos, however, enhancer N1 activity persists in the paraxial mesoderm compartment, eliciting ectopic Sox2 activation and transforming the paraxial mesoderm into neural tubes. An enhancer-N1-specific deletion mutation introduced into Tbx6 mutant embryos prevented this Sox2 activation in the mesodermal compartment and subsequent development of ectopic neural tubes, indicating that Tbx6 regulates Sox2 via enhancer N1. Tbx6-dependent repression of Wnt3a in the paraxial mesodermal compartment is implicated in this regulatory process. Paraxial mesoderm-specific misexpression of a Sox2 transgene in wild-type embryos resulted in ectopic neural tube development. Thus, Tbx6 represses Sox2 by inactivating enhancer N1 to inhibit neural development, and this is an essential step for the specification of paraxial mesoderm from the axial stem cells.
Makiko Iwafuchi-Doi, Yuzo Yoshida, Daria Onichtchouk, Manuel Leichsenring, Wolfgang Driever, Tatsuya Takemoto, Masanori Uchikawa, Yusuke Kamachi and Hisato Kondoh : The Pou5f1/Pou3f-dependent but SoxB-independent regulation of conserved enhancer N2 initiates Sox2 expression during epiblast to neural plate stages in vertebrates., Developmental Biology, Vol.352, No.2, 354-366, 2010.
(Summary)
The transcription factor Sox2 is a core component of the pluripotency control circuits in the early embryo, and later controls many aspects of neural development. Here, we demonstrate that Sox2 expression in the epiblast (mouse blastoderm) and anterior neural plate (ANP) is determined by the upstream enhancer N2. The mouse enhancer N2 exhibits strong activity in mouse ES cells, epiblast and ANP, and is regulated correctly in chicken and zebrafish embryos. Targeted deletion of this enhancer in mouse embryos caused a large reduction of Sox2 expression to 10% of that of wild-type levels in epiblast and ANP. However, this was tolerated by mouse embryo, probably due to functional compensation by Sox3. The activity of enhancer N2 depends on phylogenetically conserved bipartite POU factor-binding motifs in a 73-bp core sequence that function synergistically, but this activation does not involve Sox2. The major POU factor expressed at the epiblastic stage is Pou5f1 (Oct3/4), while those in the anterior neural plate are Pou3f factors (Oct6, Brn2 etc.). These factors are gradually exchanged during the transition from epiblast to ANP stages in mouse embryos and epiblast stem cells (EpiSC). Consistently, enhancer N2 activity changes from full Pou5f1 dependence to Pou3f dependence during the development of neural plate cells (NPC) from EpiSC, as assessed by specific POU factor knockdown in these cells. Zebrafish mutant embryos completely devoid of Pou5f1 activity failed to activate enhancer N2 and to express Sox2 in the blastoderm and ANP, and these defects were rescued by exogenous supply of pou5f1. Previously, Pou5f1-Sox2 synergism-dependent Sox2 activation through enhancer SRR2 in ES cells has been highlighted, but this mechanism is limited to ES cells and amniotes. In contrast, the enhancer N2-mediated, POU factor-dependent activation of Sox2, without involvement of Sox2, is a phylogenetically conserved core mechanism that functions in gene regulatory networks at early embryonic stages.
Yusuke Kamachi, Makiko Iwafuchi, Yuichi Okuda, Tatsuya Takemoto, Masanori Uchikawa and Hisato Kondoh : Evolution of non-coding regulatory sequences involved in the developmental process: reflection of differential employment of paralogous genes as highlighted by Sox2 and group B1 Sox genes., Proceedings of the Japan Academy. Series B, Physical and biological sciences, Vol.85, No.2, 55-68, 2009.
(Summary)
In higher vertebrates, the expression of Sox2, a group B1 Sox gene, is the hallmark of neural primordial cell state during the developmental processes from embryo to adult. Sox2 is regulated by the combined action of many enhancers with distinct spatio-temporal specificities. DNA sequences for these enhancers are conserved in a wide range of vertebrate species, corresponding to a majority of highly conserved non-coding sequences surrounding the Sox2 gene, corroborating the notion that the conservation of non-coding sequences mirrors their functional importance. Among the Sox2 enhancers, N-1 and N-2 are activated the earliest in embryogenesis and regulate Sox2 in posterior and anterior neural plates, respectively. These enhancers differ in their evolutionary history: the sequence and activity of enhancer N-2 is conserved in all vertebrate species, while enhancer N-1 is fully conserved only in amniotes. In teleost embryos, Sox19a/b play the major pan-neural role among the group B1 Sox paralogues, while strong Sox2 expression is limited to the anterior neural plate, reflecting the absence of posterior CNS-dedicated enhancers, including N-1. In Xenopus, neurally expressed SoxD is the orthologue of Sox19, but Sox3 appears to dominate other B1 paralogues. In amniotes, however, Sox19 has lost its group B1 Sox function and transforms into group G Sox15 (neofunctionalization), and Sox2 assumes the dominant position by gaining enhancer N-1 and other enhancers for posterior CNS. Thus, the gain and loss of specific enhancer elements during the evolutionary process reflects the change in functional assignment of particular paralogous genes, while overall regulatory functions attributed to the gene family are maintained.
Kazunobu Moriguchi, Tatsuya Takemoto, Takafumi Aoki, Shin-ichi Nakakita, Shunji Natsuka and Sumihiro Hase : Free oligosaccharides with Lewis x structure expressed in the segmentation period of zebrafish embryo., The Journal of Biochemistry, Vol.142, No.2, 213-227, 2007.
(Summary)
We previously reported that zebrafishalpha1-3fucosyltrasferase 1 (zFT1) was expressed in embryos at the segmentation period, and was capable of synthesizing the Lewis x epitope [Gal beta1-4(Fuc alpha1-3)GlcNAc] [Kageyama et.al, J. Biochem., 125, 838-845 (1999)]. In the current study, we attempted to detect the enzyme products of zFT1 in zebrafish embryos. Oligosaccharides were prepared from the zebrafish embryos at 12, 18 and 48 h after fertilization and labelled with a fluorophore, 2-aminopyridine, for highly sensitive detections. Pyridylamino (PA)-oligosaccharides that were alpha1-3/4fucosidase sensitive and time-dependently expressed at 18 h after fertilization were identified as candidates for the in vivo products synthesized by zFT1. Structures of these oligosaccharides were determined by a combination of exoglycosidase digestions and two-dimensional HPLC sugar mapping to be the biantennary complex-type structures with two Lewis x epitopes: (Gal beta1-4)(0,1,2)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]}Man beta1-4GlcNAc, and (Gal beta1-4)(0,1)-{Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-6[Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-2Man alpha1-3]} Man beta1-4GlcNAc beta1-4GlcNAc. The presence of Lewis x structure of these oligosaccharides together with their expression time suggests that they are products of zFT1. Remarkably, most of these oligosaccharides were free form. Furthermore, we detected an endo-beta-N-acetylglucosaminidase activity in the 18 h embryo. These results suggest that the oligosaccharides synthesized by zFT1 are present in the embryo at the segmentation period in free form, owing to the liberation from glycoproteins with endo-beta-N-acetylglucosaminidase(s) and/or glycoamidase(s).
Tatsuya Takemoto, Masanori Uchikawa, Yusuke Kamachi and Hisato Kondoh : Convergence of Wnt and FGF signals in the genesis of posterior neural plate through activation of the Sox2 enhancer N-1., Development, Vol.133, No.2, 297-306, 2005.
(Summary)
The expression of the transcription factor gene Sox2 precisely marks the neural plate in various vertebrate species. We previously showed that the Sox2 expression prevailing in the neural plate of chicken embryos is actually regulated by the coordination of five phylogenetically conserved enhancers having discrete regional coverage, among which the 420-bp long enhancer N-1, active in the node-proximal region, is probably involved directly in the genesis of the posterior neural plate. We investigated the signaling systems regulating this enhancer, first identifying the 56-bp N-1 core enhancer (N-1c), which in a trimeric form recapitulates the activity of the enhancer N-1. Mutational analysis identified five blocks, A to E, that regulate the enhancer N-1c. Functional analysis of these blocks indicated that Wnt and FGF signals synergistically activate the enhancer through Blocks A-B, bound by Lef1, and Block D, respectively. Fgf8b and Wnt8c expressed in the organizer-primitive streak region account for the activity in the embryo. Block E is essential for the repression of the enhancer N-1c activity in the mesendodermal precursors. The enhancer N-1c is not affected by BMP signals. Thus, Wnt and FGF signals converge to activate Sox2 expression through the enhancer N-1c, revealing the direct involvement of the Wnt signal in the initiation of neural plate development.
Tatsuya Takemoto, Shunji Natsuka, Shin-Ichi Nakakita and Sumihiro Hase : Expression of complex-type N-glycans in developmental periods of zebrafish embryo., Glycoconjugate Journal, Vol.22, No.1-2, 21-26, 2005.
(Summary)
As a first step to elucidate a role of N-glycans in development of vertebrates, we analyzed structures of the glycans expressed in early stages of zebrafish embryo. N-glycans were prepared from zebrafish embryos at several developmental stages followed by tagging with a fluorophore, 2-aminopyridine. The labeled glycans were analyzed by two modes of HPLC's. The comparison of the elution profiles of HPLC's unveil the change of the oligosaccharide structure during the development. These peaks were merely detected during 4-7 h after fertilization, however, increased from 12 h, and at 15 h a fairly amount of them was appeared. Structure analysis revealed that they were bianntenary complex-type N-glycans with or without fucose and/or bisecting N-acetylglucosamine residues. These results suggest that the complex-type N-glycans are concerned in some developmental event from segmentation period downward in zebrafish.
Masanori Uchikawa, Tatsuya Takemoto, Yusuke Kamachi and Hisato Kondoh : Efficient identification of regulatory sequences in the chicken genome by a powerful combination of embryo electroporation and genome comparison., Mechanisms of Development, Vol.121, No.9, 1145-1158, 2004.
(Summary)
Recently expanded knowledge of gene regulation clearly indicates that the regulatory sequences of a gene, usually identified as enhancers, are widely distributed in the gene locus, revising the classical view that they are clustered in the vicinity of genes. To identify regulatory sequences for Sox2 expression governing early neurogenesis, we scanned the 50-kb region of the chicken Sox2 locus for enhancer activity utilizing embryo electroporation, resulting in identification of a number of enhancers scattered throughout the analyzed genomic span. The 'pan-neural' Sox2 expression in early embryos is actually brought about by the composite activities of five separate enhancers with distinct spatio-temporal specificities. These and other functionally defined enhancers exactly correspond to extragenic sequence blocks that are conspicuously conserved between the chicken and mammalian genomes and that are embedded in sequences with a wide range of sequence conservation between humans and mice. The sequences conserved between amniotes and teleosts correspond to subregions of the enhancer subsets which presumably represent core motifs of the enhancers, and the limited conservation partly reflects divergent expression patterns of the gene. The phylogenic distance between the chicken and mammals appears optimal for identifying a battery of genetic regulatory elements as conserved sequence blocks, and chicken embryo electroporation facilitates functional characterization of these elements.
Masanori Uchikawa, Yoshiko Ishida, Tatsuya Takemoto, Yusuke Kamachi and Hisato Kondoh : Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals., Developmental Cell, Vol.4, No.4, 509-519, 2003.
(Summary)
Sox2 expression marks neural and sensory primordia at various stages of development. A 50 kb genomic region of chicken Sox2 was isolated and scanned for enhancer activity utilizing embryo electroporation, resulting in identification of a battery of enhancers. Although Sox2 expression in the early embryonic CNS appears uniform, it is actually pieced together by five separate enhancers with distinct spatio-temporal specificities, including the one activated by the neural induction signals emanating from Hensen's node. Enhancers for Sox2 expression in the lens and nasal/otic placodes and in the neural crest were also determined. These functionally identified Sox2 enhancers exactly correspond to the extragenic sequence blocks conspicuously conserved between chicken and mammals, which are not discernible by sequence comparison among mammals.
(Link to Search Site for Scientific Articles)
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Fuminori Tanihara, LTK Do, TV Nguyen, Toshiki Kunihara, Katsutoshi Nishio, Tatsuya Takemoto and Takeshige Otoi : Generation of TP53-modified pigs by GEEP method: CRISPR/Cas9-mediated gene modification introduced into porcine zygotes by electroporation., Transgenic Research, Vol.26, No.1, 38, Utah, USA, Oct. 2017.
2.
Sayumi Fujimori, Izumi Ohigashi, Tatsuya Takemoto, Yousuke Takahama and Takada Shinji : Activation of Wnt/β-catenin signaling in thymic epithelial progenitors, 7th International Workshop of Kyoto T Cell Conference, Kyoto, Mar. 2017.
3.
Izumi Ohigashi, Uddin Myn, Tatsuya Takemoto and Yousuke Takahama : Foxn1-binding cis-regulatory element required for optimal CD8+ T cell production in the thymus, 11th International Symposium of The Institute Network Frontiers in Biomedical Sciences, Jan. 2017.
4.
Tatsuya Takemoto : High-throughput production of mutant mice by electroporation of CRISPR/Cas9 system, Mouse Molecular Genetics 2015, Sep. 2015.
Eisuke Shimokita, Hitomi Suzuki, Yoshihiro Tsuruo and Tatsuya Takemoto : Mesodermal cell migration does NOT depend on mesoderm-inducible transcriptional factor cTbx6L, 第52回日本発生生物学会, May 2019.
Tatsuya Takemoto : High-throughput production of mutant mice by electroporation of CRISPR/Cas9 system, 48th Annual Meeting of the Japanese Society Biologists, Jun. 2015.
Tatsuya Takemoto : Frontiers in Developmental Biology by Unique Approaches, 47th Annual Meeting of the Japanese Society of Developmental Biologists, May 2014.
20.
Tatsuya Takemoto and Kondoh Hisato : The role of Tbx6 in the derivation of mesodermal tissue from the axial stem cells, 47th Annual Meeting of the Japanese Society of Developmental Biologists, May 2014.
Et cetera, Workshop:
1.
Fuminori Tanihara, Maki Hirata, Nguyen Thi Nhien, Le Anh Quynh, Takayuki Hirano, Tatsuya Takemoto, 中井 美智子, 淵本 大一郎 and Takeshige Otoi : ゲノム編集によるTP53遺伝子改変ブタの作製と表現型の解析, 第6回日本先進医工学ブタ研究会, Oct. 2018.
2.
Fuminori Tanihara, Tatsuya Takemoto, Maki Hirata, N Nguyen Thi, Toshiki Kunihara, R Nishinakamura and Takeshige Otoi : Modification of SALL1 gene via CRISPR/Cas9-mediated gene editing introduced into porcine zygotes by electroporation, KEY Forum: The 3rd International symposium on Stem Cell Traits and Developmental Systems, Jan. 2018.
3.
Fuminori Tanihara, Tatsuya Takemoto, Michiko Nakai, Eri Kitagawa, Do Thi Kim Lanh, Akira Onishi, Shunichi Suzuki, Syoichiro Senbon, Daiichiro Fuchimoto and Takeshige Otoi : 新規ゲノム編集技術を用いたPDX-1遺伝子改変ブタの作製, 第4回 日本先進医工学ブタ研究会 要旨集, 21, Oct. 2016.
Functional analysis of the cell initiating gastrulation process. (Project/Area Number: 21K19270 )
Identification and functional analysis of noncoding SNPs causative for genetic disorders (Project/Area Number: 18KT0024 )
Analysis of the singularity cells controlling the pattern formation of multicellular system (Project/Area Number: 18H05415 )
Production of pigs for safe xenotransplantation by controlling infectious porcine endogenous retrovirus (PERV) (Project/Area Number: 17K19325 )
Production of genetically modified pigs by combined techniques of chromosome engineering and genome editing (Project/Area Number: 17H03938 )
Regularion of axial stem cells producing various types of somatic cells (Project/Area Number: 17H03607 )
Innovation of novel therapeutics against diabetic nephropathy targeting ChREBP (Project/Area Number: 16K15492 )
Generation of reporter mice by electroporating genome editing system into mouse zygotes (Project/Area Number: 16K14738 )
Cell lineage analysis of mouse embryos (Project/Area Number: 26670092 )
Gene regulatory network underlying epiblast cell determination and their derivation into various somatic cell lineages (Project/Area Number: 26251024 )