Kyushu University Academic Staff Educational and Research Activities Database
List of Papers
hayashi katsuhiko Last modified date:2021.07.13

Professor / Department of Stem Cell Biology and Medicine / Faculty of Medical Sciences


Papers
1. Katsuhiko Hayashi, So Shimamoto, Go Nagamatsu, Environmental factors for establishment of the dormant state in oocytes, Development Growth and Differentiation, 10.1111/dgd.12653, 62, 3, 150-157, 2020.04, Guaranteeing the sustainability of gametogenesis is a fundamental issue for perpetuating the species. In the mammalian ovary, sustainability is accomplished by keeping a number of oocytes “stocked” in the dormant state. Despite the evident importance of this state, the mechanisms underlying the oocyte dormancy are not fully understood, although it is presumed that both intrinsic and extrinsic factors are involved. Here, we review environmental factors involved in the regulation of oocyte dormancy. Consideration of the environmental factors illustrates the nature of the ovarian compartment, in which primordial follicles reside. This should greatly improve our understanding of the mechanisms and also assist in reconstitution of the dormant state in culture. Accumulating knowledge on the dormant state of oocytes will contribute to a wide range of research in fields such as developmental biology, reproductive biology and regenerative medicine..
2. Norio Hamada, Nobuhiko Hamazaki, So Shimamoto, Orie Hikabe, Go Nagamatsu, Yuki Takada, Kiyoko Kato, Katsuhiko Hayashi, Germ cell-intrinsic effects of sex chromosomes on early oocyte differentiation in mice, PLoS genetics, 10.1371/journal.pgen.1008676, 16, 3, 2020.03, A set of sex chromosomes is required for gametogenesis in both males and females, as represented by sex chromosome disorders causing agametic phenotypes. Although studies using model animals have investigated the functional requirement of sex chromosomes, involvement of these chromosomes in gametogenesis remains elusive. Here, we elicit a germ cell-intrinsic effect of sex chromosomes on oogenesis, using a novel culture system in which oocytes were induced from embryonic stem cells (ESCs) harboring XX, XO or XY. In the culture system, oogenesis using XO and XY ESCs was severely disturbed, with XY ESCs being more strongly affected. The culture system revealed multiple defects in the oogenesis of XO and XY ESCs, such as delayed meiotic entry and progression, and mispairing of the homologous chromosomes. Interestingly, Eif2s3y, a Y-linked gene that promotes proliferation of spermatogonia, had an inhibitory effect on oogenesis. This led us to the concept that male and female gametogenesis appear to be in mutual conflict at an early stage. This study provides a deeper understanding of oogenesis under a sex-reversal condition..
3. So I. Nagaoka, Fumio Nakaki, Hidetaka Miyauchi, Yoshiaki Nosaka, Hiroshi Ohta, Yukihiro Yabuta, Kazuki Kurimoto, Katsuhiko Hayashi, Tomonori Nakamura, Takuya Yamamoto, Mitinori Saitou, ZGLP1 is a determinant for the oogenic fate in mice, Science, 10.1126/science.aay5947, 367, 6482, 2020.03, Sex determination of germ cells is vital to creating the sexual dichotomy of germ cell development, thereby ensuring sexual reproduction. However, the underlying mechanisms remain unclear. Here, we show that ZGLP1, a conserved transcriptional regulator with GATA-like zinc fingers, determines the oogenic fate inmice. ZGLP1 acts downstream of bone morphogenetic protein, but not retinoic acid (RA), and is essential for the oogenic program and meiotic entry. ZGLP1 overexpression induces differentiation of in vitro primordial germ cell-like cells (PGCLCs) into fetal oocytes by activating the oogenic programs repressed by Polycomb activities, whereas RA signaling contributes to oogenic program maturation and PGC program repression. Our findings elucidate the mechanism for mammalian oogenic fate determination, providing a foundation for promoting in vitro gametogenesis and reproductive medicine..
4. So I. Nagaoka, Fumio Nakaki, Hidetaka Miyauchi, Yoshiaki Nosaka, Hiroshi Ohta, Yukihiro Yabuta, Kazuki Kurimoto, Katsuhiko Hayashi, Tomonori Nakamura, Takuya Yamamoto, Mitinori Saitou, ZGLP1 is a determinant for the oogenic fate in mice, Science (New York, N.Y.), 10.1126/science.aaw4115, 367, 6482, 2020.03, Sex determination of germ cells is vital to creating the sexual dichotomy of germ cell development, thereby ensuring sexual reproduction. However, the underlying mechanisms remain unclear. Here, we show that ZGLP1, a conserved transcriptional regulator with GATA-like zinc fingers, determines the oogenic fate in mice. ZGLP1 acts downstream of bone morphogenetic protein, but not retinoic acid (RA), and is essential for the oogenic program and meiotic entry. ZGLP1 overexpression induces differentiation of in vitro primordial germ cell-like cells (PGCLCs) into fetal oocytes by activating the oogenic programs repressed by Polycomb activities, whereas RA signaling contributes to oogenic program maturation and PGC program repression. Our findings elucidate the mechanism for mammalian oogenic fate determination, providing a foundation for promoting in vitro gametogenesis and reproductive medicine..
5. So I. Nagaoka, Fumio Nakaki, Hidetaka Miyauchi, Yoshiaki Nosaka, Hiroshi Ohta, Yukihiro Yabuta, Kazuki Kurimoto, Katsuhiko Hayashi, Tomonori Nakamura, Takuya Yamamoto, Mitinori Saitou, ZGLP1 is a determinant for the oogenic fate in mice, Science (New York, N.Y.), 10.1126/science.aaw4115, 367, 6482, 2020.03, Sex determination of germ cells is vital to creating the sexual dichotomy of germ cell development, thereby ensuring sexual reproduction. However, the underlying mechanisms remain unclear. Here, we show that ZGLP1, a conserved transcriptional regulator with GATA-like zinc fingers, determines the oogenic fate in mice. ZGLP1 acts downstream of bone morphogenetic protein, but not retinoic acid (RA), and is essential for the oogenic program and meiotic entry. ZGLP1 overexpression induces differentiation of in vitro primordial germ cell-like cells (PGCLCs) into fetal oocytes by activating the oogenic programs repressed by Polycomb activities, whereas RA signaling contributes to oogenic program maturation and PGC program repression. Our findings elucidate the mechanism for mammalian oogenic fate determination, providing a foundation for promoting in vitro gametogenesis and reproductive medicine..
6. Maiko Terada, Masaki Kawamata, Ryota Kimura, Sayaka Sekiya, Go Nagamatsu, Katsuhiko Hayashi, Kenichi Horisawa, Atsushi Suzuki, Generation of Nanog reporter mice that distinguish pluripotent stem cells from unipotent primordial germ cells, Genesis, 10.1002/dvg.23334, 57, 11-12, 2019.11, Nanog is a core transcription factor specifically expressed not only in the pluripotent stem cells (PSCs), such as embryonic stem cells (ESCs), embryonic germ cells (EGCs), and induced PSCs (iPSCs), but also in the unipotent primordial germ cells (PGCs). Although Nanog promoter/enhancer regions are well characterized by in vitro analyses, direct correlations between the regulatory elements for Nanog expression and in vivo expression patterns of Nanog have not been fully clarified. In this study, we generated Nanog-RFP transgenic (Tg) mice in which expression of red fluorescent protein (RFP) is driven by a 5.2 kb Nanog promoter/enhancer region. As expected, RFP was expressed in the inner cell mass of blastocysts, ESCs, and iPSCs. However, RFP fluorescence was not observed in PGCs, although Nanog was expressed in PGCs. Because RFP fluorescence was visible in the PGC-derived pluripotent EGCs in culture, it was suggested that the reporter gene expression was specifically activated in PSCs. In conclusion, we have generated a novel Nanog-RFP Tg mouse line that can selectively tag PSCs over unipotent PGCs..
7. Katsuhiko Hayashi, In vitro reconstitution of germ cell development, Biology of reproduction, 10.1093/biolre/ioz111, 101, 3, 567-578, 2019.09, Germ cell development is a series of highly specialized processes through which diploid pluripotent cells differentiate into haploid gametes. The processes include biologically important events such as epigenetic reprogramming, sex determination, and meiosis. The mechanisms underlying these events are key issues in reproductive and developmental biology, yet they still remain elusive. As a tool to elucidate these mechanisms, in vitro gametogenesis, which reproduces germ cell development in culture, has long been sought for decades. Recently, methods of in vitro gametogenesis have undergone rapid development in association with stem cell biology, opening many possibilities in this field. This new technology is considered an alternative source of gametes for the reproduction of animals and perhaps humans. This review summarizes current advances and problems in in vitro gametogenesis..
8. So Shimamoto, Yohei Nishimura, Go Nagamatsu, Norio Hamada, Haruka Kita, Orie Hikabe, Nobuhiko Hamazaki, Katsuhiko Hayashi, Hypoxia induces the dormant state in oocytes through expression of Foxo3, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1817223116, 116, 25, 12321-12326, 2019.06, In mammals, most immature oocytes remain dormant in the primordial follicles to ensure the longevity of female reproductive life. A precise understanding of mechanisms underlying the dormancy is important for reproductive biology and medicine. In this study, by comparing mouse oogenesis in vivo and in vitro, the latter of which bypasses the primordial follicle stage, we defined the gene-expression profile representing the dormant state of oocytes. Overexpression of constitutively active FOXO3 partially reproduced the dormant state in vitro. Based on further gene-expression analysis, we found that a hypoxic condition efficiently induced the dormant state in vitro. The effect of hypoxia was severely diminished by disruption of the Foxo3 gene and inhibition of hypoxia-inducible factors. Our findings provide insights into the importance of environmental conditions and their effectors for establishing the dormant state..
9. Eli Y. Adashi, I. Glenn Cohen, Jacob H. Hanna, Azim M. Surani, Katsuhiko Hayashi, Stem Cell-Derived Human Gametes
The Public Engagement Imperative, Trends in Molecular Medicine, 10.1016/j.molmed.2019.01.005, 25, 3, 165-167, 2019.03, The implications of scientific breakthroughs are rarely faced up to in advance of their realization. Stem cell-derived human gametes, a disruptive technology in waiting, are likely to recapitulate this historic pattern absent active intervention. Herein we call for the conduct of thoughtful ante hoc deliberations on the prospect of stem cell-derived human gametes with an eye toward minimizing potential untoward post hoc regulatory or statutory impositions..
10. Go Nagamatsu, So Shimamoto, Nobuhiko Hamazaki, Yohei Nishimura, Katsuhiko Hayashi, Mechanical stress accompanied with nuclear rotation is involved in the dormant state of mouse oocytes, Science Advances, 10.1126/sciadv.aav9960, 5, 6, 2019, The most immature oocytes remain dormant in primordial follicles in the ovary, ensuring the longevity of female reproductive life. Despite its biological and clinical importance, knowledge of mechanisms regulating the dormant state remains limited. Here, we show that mechanical stress plays a key role in maintaining the dormant state of the oocytes in primordial follicles in mice. Transcriptional and histological analyses revealed that oocytes were compressed by surrounding granulosa cells with extracellular matrix. This environmental state is functionally crucial, as oocytes became activated upon loosening the structure and the dormancy was restored by additional compression with exogenous pressure. The nuclei of oocytes in primordial follicles rotated in response to the mechanical stress. Pausing the rotation triggered activation of oocytes through nuclear export of forkhead box O3 (FOXO3). These results provide insights into the mechanisms by which oocytes are kept dormant to sustain female reproductive life..
11. Thomas B. Hildebrandt, Robert Hermes, Silvia Colleoni, Sebastian Diecke, Susanne Holtze, Marilyn B. Renfree, Jan Stejskal, Katsuhiko Hayashi, Micha Drukker, Pasqualino Loi, Frank Göritz, Giovanna Lazzari, Cesare Galli, Embryos and embryonic stem cells from the white rhinoceros, Nature communications, 10.1038/s41467-018-04959-2, 9, 1, 2018.12, The northern white rhinoceros (NWR, Ceratotherium simum cottoni) is the most endangered mammal in the world with only two females surviving. Here we adapt existing assisted reproduction techniques (ART) to fertilize Southern White Rhinoceros (SWR) oocytes with NWR spermatozoa. We show that rhinoceros oocytes can be repeatedly recovered from live SWR females by transrectal ovum pick-up, matured, fertilized by intracytoplasmic sperm injection and developed to the blastocyst stage in vitro. Next, we generate hybrid rhinoceros embryos in vitro using gametes of NWR and SWR. We also establish embryonic stem cell lines from the SWR blastocysts. Blastocysts are cryopreserved for later embryo transfer. Our results indicate that ART could be a viable strategy to rescue genes from the iconic, almost extinct, northern white rhinoceros and may also have broader impact if applied with similar success to other endangered large mammalian species..
12. Katsuhiko Hayashi, Current advance of in vitro gametogenesis, Seikagaku, 10.14952/SEIKAGAKU.2018.900533, 90, 4, 533-538, 2018.01.
13. Katsuhiko Hayashi, Oogenesis in vitro, Encyclopedia of Reproduction, 10.1016/B978-0-12-801238-3.64458-6, 225-231, 2018.01, Reconstitution of oogenesis in vitro provides a useful tool for improving our understanding of oogenesis and also an alternative source of oocytes for reproduction. In mice, it has become possible to produce fully mature oocytes from pluripotent stem cells (PSCs) in culture. The PSC-derived oocytes fertilized with wild-type sperm are capable of giving rise to pups that grow to fertile adult mice. This section describes how to reconstitute oogenesis in vitro including technical backgrounds, similarity and difference between in vivo and in vitro oogenesis, and a current situation of the application to humans..
14. Go Nagamatsu, Katsuhiko Hayashi, Stem cells, in vitro gametogenesis and male fertility, Reproduction (Cambridge, England), 10.1530/REP-17-0510, 154, 6, F79-F91, 2017.12, Reconstitution in culture of biological processes, such as differentiation and organization, is a key challenge in regenerative medicine, and one in which stem cell technology plays a central role. Pluripotent stem cells and spermatogonial stem cells are useful materials for reconstitution of germ cell development in vitro, as they are capable of differentiating into gametes. Reconstitution of germ cell development, termed in vitro gametogenesis, will provide an experimental platform for a better understanding of germ cell development, as well as an alternative source of gametes for reproduction, with the potential to cure infertility. Since germ cells are the cells for 'the next generation', both the culture system and its products must be carefully evaluated. In this issue, we summarize the progress in in vitro gametogenesis, most of which has been made using mouse models, as well as the future challenges in this field..
15. Miyauchi H, Ohta H, Nagaoka S, Nakaki F, Sasaki K, Hayashi K, Yabuta Y, Nakamura T, Yamamoto T, Saitou M., Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice., EMBO J, 2017.11, The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells in vitro under a defined condition without the use of gonadal somatic cells. We show that retinoic acid (RA) and its key effector, STRA8, are not sufficient to induce the female germ-cell fate. In contrast, bone morphogenetic protein (BMP) and RA synergistically induce primordial germ cells (PGCs)/PGC-like cells (PGCLCs) derived from embryonic stem cells (ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression in vivo faithfully. Importantly, the female germ-cell induction necessitates a proper cellular competence-most typically, DNA demethylation of relevant genes-which is observed in appropriately propagated PGCs/PGCLCs, but not in PGCs/PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ-cell induction. Our findings represent a framework for a comprehensive delineation of the sex-determination pathway in mammalian germ cells, including humans..
16. Hidetaka Miyauchi, Hiroshi Ohta, So Nagaoka, Fumio Nakaki, Kotaro Sasaki, Katsuhiko Hayashi, Yukihiro Yabuta, Tomonori Nakamura, Takuya Yamamoto, Mitinori Saitou, Bone morphogenetic protein and retinoic acid synergistically specify female germ-cell fate in mice, EMBO Journal, 10.15252/embj.201796875, 36, 21, 3100-3119, 2017.11, The mechanism for sex determination in mammalian germ cells remains unclear. Here, we reconstitute the female sex determination in mouse germ cells in vitro under a defined condition without the use of gonadal somatic cells. We show that retinoic acid (RA) and its key effector, STRA8, are not sufficient to induce the female germ-cell fate. In contrast, bone morphogenetic protein (BMP) and RA synergistically induce primordial germ cells (PGCs)/PGC-like cells (PGCLCs) derived from embryonic stem cells (ESCs) into fetal primary oocytes. The induction is characterized by entry into the meiotic prophase, occurs synchronously and recapitulates cytological and transcriptome progression in vivo faithfully. Importantly, the female germ-cell induction necessitates a proper cellular competence—most typically, DNA demethylation of relevant genes—which is observed in appropriately propagated PGCs/PGCLCs, but not in PGCs/PGCLCs immediately after induction. This provides an explanation for the differential function of BMP signaling between PGC specification and female germ-cell induction. Our findings represent a framework for a comprehensive delineation of the sex-determination pathway in mammalian germ cells, including humans..
17. Katsuhiko Hayashi, Orie Hikabe, Yayoi Obata, Yuji Hirao, Reconstitution of mouse oogenesis in a dish from pluripotent stem cells, Nature Protocols, 10.1038/nprot.2017.070, 12, 9, 1733-1744, 2017.09, Generation of functional oocytes in culture from pluripotent stem cells should provide a useful model system for improving our understanding of the basic mechanisms underlying oogenesis. In addition, it has potential applications as an alternative source of oocytes for reproduction. Using the most advanced mouse model in regard to reproductive engineering and stem cell biology, we previously developed a culture method that produces functional primorial germ cells starting from pluripotent cells in culture and described it in a previous protocol. This Protocol Extension describes an adaptation of this existing Protocol in which oogenesis also occurs in vitro, thus substantially modifying the technique. Oocytes generated from embryonic stem cells (ESCs) or induced pluripotent stem cells give rise to healthy pups. Here, we describe the protocol for oocyte generation in culture. The protocol is mainly composed of three different culture stages: in vitro differentiation (IVDi), in vitro growth (IVG), and in vitro maturation (IVM), which in total take ∼5 weeks. In each culture period, there are several checkpoints that enable the number of oocytes being produced in the culture to be monitored. The basic structure of the culture system should provide a useful tool for clarifying the complicated sequence of oogenesis in mammals..
18. Mitsuhiro Endoh, Takaho A. Endo, Jun Shinga, Katsuhiko Hayashi, Anca Farcas, Kit Wan Ma, Shinsuke Ito, Jafar Sharif, Tamie Endoh, Naoko Onaga, Manabu Nakayama, Tomoyuki Ishikura, Osamu Masui, Benedikt M. Kessler, Toshio Suda, Osamu Ohara, Akihiko Okuda, Robert J. Klose, Haruhiko Koseki, Erratum
Correction: PCGF6-PRC1 suppresses premature differentiation of mouse embryonic stem cells by regulating germ cell-related genes (eLife (2017) 6 PII: e27970), eLife, 10.7554/eLife.27970, 6, 2017.04.
19. Endoh M, Endo TA, Shinga J, Hayashi K, Farcas A, Ma KW, Ito S, Sharif J, Endoh T, Onaga N, Nakayama M, Ishikura T, Masui O, Kessler BM, Suda T, Ohara O, Okuda A, Klose R, Koseki H., PCGF6-PRC1 suppresses premature differentiation of mouse embryonic stem cells by regulating germ cell-related genes., Elife, doi: 10.7554/eLife.21064., 2017.03, The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of Pcgf6 in ESCs leads to robust de-repression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells..
20. Mitsuhiro Endoh, Takaho A. Endo, Jun Shinga, Katsuhiko Hayashi, Anca Farcas, Kit Wan Ma, Shinsuke Ito, Jafar Sharif, Tamie Endoh, Naoko Onaga, Manabu Nakayama, Tomoyuki Ishikura, Osamu Masui, Benedikt M. Kessler, Toshio Suda, Osamu Ohara, Akihiko Okuda, Robert Klose, Haruhiko Koseki, PCGF6-PRC1 suppresses premature differentiation of mouse embryonic stem cells by regulating germ cell-related genes, eLife, 10.7554/eLife.21064, 6, 2017.03, The ring finger protein PCGF6 (polycomb group ring finger 6) interacts with RING1A/B and E2F6 associated factors to form a non-canonical PRC1 (polycomb repressive complex 1) known as PCGF6-PRC1. Here, we demonstrate that PCGF6-PRC1 plays a role in repressing a subset of PRC1 target genes by recruiting RING1B and mediating downstream mono-ubiquitination of histone H2A. PCGF6-PRC1 bound loci are highly enriched for promoters of germ cell-related genes in mouse embryonic stem cells (ESCs). Conditional ablation of Pcgf6 in ESCs leads to robust derepression of such germ cell-related genes, in turn affecting cell growth and viability. We also find a role for PCGF6 in pre- and peri-implantation mouse embryonic development. We further show that a heterodimer of the transcription factors MAX and MGA recruits PCGF6 to target loci. PCGF6 thus links sequence specific target recognition by the MAX/MGA complex to PRC1-dependent transcriptional silencing of germ cell-specific genes in pluripotent stem cells..
21. Hidehiro Toh, Kenjiro Shirane, Fumihito Miura, Naoki Kubo, Kenji Ichiyanagi, Katsuhiko Hayashi, Mitinori Saitou, Mikita Suyama, Takashi Ito, Hiroyuki Sasaki, Software updates in the illumina hiseq platform affect whole-genome bisulfite sequencing, BMC genomics, 10.1186/s12864-016-3392-9, 18, 1, 2017.01, Background: Methylation of cytosine in genomic DNA is a well-characterized epigenetic modification involved in many cellular processes and diseases. Whole-genome bisulfite sequencing (WGBS), such as MethylC-seq and post-bisulfite adaptor tagging sequencing (PBAT-seq), uses the power of high-throughput DNA sequencers and provides genome-wide DNA methylation profiles at single-base resolution. However, the accuracy and consistency of WGBS outputs in relation to the operating conditions of high-throughput sequencers have not been explored. Results: We have used the Illumina HiSeq platform for our PBAT-based WGBS, and found that different versions of HiSeq Control Software (HCS) and Real-Time Analysis (RTA) installed on the system provided different global CpG methylation levels (approximately 5% overall difference) for the same libraries. This problem was reproduced multiple times with different WGBS libraries and likely to be associated with the low sequence diversity of bisulfite-converted DNA. We found that HCS was the major determinant in the observed differences. To determine which version of HCS is most suitable for WGBS, we used substrates with predetermined CpG methylation levels, and found that HCS v2.0.5 is the best among the examined versions. HCS v2.0.12 showed the poorest performance and provided artificially lower CpG methylation levels when 5-methylcytosine is read as guanine (first read of PBAT-seq and second read of MethylC-seq). In addition, paired-end sequencing of low diversity libraries using HCS v2.2.38 or the latest HCS v2.2.58 was greatly affected by cluster densities. Conclusions: Software updates in the Illumina HiSeq platform can affect the outputs from low-diversity sequencing libraries such as WGBS libraries. More recent versions are not necessarily the better, and HCS v2.0.5 is currently the best for WGBS among the examined HCS versions. Thus, together with other experimental conditions, special care has to be taken on this point when CpG methylation levels are to be compared between different samples by WGBS..
22. Nobuhiko Hamazaki, Kinichi Nakashima, Katsuhiko Hayashi, Takuya Imamura, Detection of bidirectional promoter-derived lncRNAs from small-scale samples using pre-amplification-free directional RNA-seq method, Methods in Molecular Biology, 10.1007/978-1-4939-6988-3_6, 83-103, 2017, Development of high-throughput sequencing technologies has uncovered the immensity of the long noncoding RNA (lncRNA) world. Divergently transcribed lncRNAs from bidirectional gene promoters, called promoter-associated noncoding RNAs (pancRNAs), account for ~20% of the total number of lncRNAs, and this major fraction is involved in many biological processes, such as development and cancer formation. Recently, we have found that the pancRNAs activate their partner genes, as represented by the fact that pancIl17d, a pancRNA that is transcribed from the antisense strand of the promoter region of Interleukin 17d (Il17d) at the onset of zygotic gene activation (ZGA), is essential for mouse preimplantation development through Il17d upregulation. The discovery of the expression of a specific set of pancRNAs during ZGA was achieved by using a method that generates directional RNA-seq libraries from small-scale samples. Although there are several methods available for small-scale samples, most of them require a pre-amplification procedure that frequently generates some amplification biases toward a subset of transcripts. We provide here a highly sensitive and reproducible method based on the preparation of directional RNA-seq libraries from as little as 100 mouse oocytes or embryos without pre-amplification for the quantification of lncRNAs as well as mRNAs..
23. Katsuhiko Hayashi, Anthony Perry, Redefining reproduction, Biochemist, 10.1042/bio03901034, 39, 1, 34-37, 2017.
24. Ishikura Y, Yabuta Y, Ohta H, Hayashi K, Nakamura T, Okamoto I, Yamamoto T, Kurimoto K, Shirane K, Sasaki H, Saitou M., In Vitro Derivation and Propagation of Spermatogonial Stem Cell Activity from Mouse Pluripotent Stem Cells., Cell Rep. , doi: 10.1016/j.celrep.2016.11.026., 2016.12, The in vitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells in vitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the in vitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation..
25. Yukiko Ishikura, Yukihiro Yabuta, Hiroshi Ohta, Katsuhiko Hayashi, Tomonori Nakamura, Ikuhiro Okamoto, Takuya Yamamoto, Kazuki Kurimoto, Kenjiro Shirane, Hiroyuki Sasaki, Mitinori Saitou, In Vitro Derivation and Propagation of Spermatogonial Stem Cell Activity from Mouse Pluripotent Stem Cells, Cell Reports, 10.1016/j.celrep.2016.11.026, 17, 10, 2789-2804, 2016.12, The in vitro derivation and propagation of spermatogonial stem cells (SSCs) from pluripotent stem cells (PSCs) is a key goal in reproductive science. We show here that when aggregated with embryonic testicular somatic cells (reconstituted testes), primordial germ cell-like cells (PGCLCs) induced from mouse embryonic stem cells differentiate into spermatogonia-like cells in vitro and are expandable as cells that resemble germline stem cells (GSCs), a primary cell line with SSC activity. Remarkably, GSC-like cells (GSCLCs), but not PGCLCs, colonize adult testes and, albeit less effectively than GSCs, contribute to spermatogenesis and fertile offspring. Whole-genome analyses reveal that GSCLCs exhibit aberrant methylation at vulnerable regulatory elements, including those critical for spermatogenesis, which may restrain their spermatogenic potential. Our study establishes a strategy for the in vitro derivation of SSC activity from PSCs, which, we propose, relies on faithful epigenomic regulation..
26. Hikabe O, Hamazaki N, Nagamatsu G, Obata Y, Hirao Y, Hamada N, Shimamoto S, Imamura T, Nakashima K, Saitou M, Hayashi K., Reconstitution in vitro of the entire cycle of the mouse female germ line., Nature, doi: 10.1038/nature20104. , 2016.11, The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg. The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro, thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture..
27. Orie Hikabe, Nobuhiko Hamazaki, Go Nagamatsu, Yayoi Obata, Yuji Hirao, Norio Hamada, So Shimamoto, Takuya Imamura, Kinichi Nakashima, Mitinori Saitou, Katsuhiko Hayashi, Reconstitution in vitro of the entire cycle of the mouse female germ line, Nature, 10.1038/nature20104, 539, 7628, 299-303, 2016.11, The female germ line undergoes a unique sequence of differentiation processes that confers totipotency to the egg. The reconstitution of these events in vitro using pluripotent stem cells is a key achievement in reproductive biology and regenerative medicine. Here we report successful reconstitution in vitro of the entire process of oogenesis from mouse pluripotent stem cells. Fully potent mature oocytes were generated in culture from embryonic stem cells and from induced pluripotent stem cells derived from both embryonic fibroblasts and adult tail tip fibroblasts. Moreover, pluripotent stem cell lines were re-derived from the eggs that were generated in vitro, thereby reconstituting the full female germline cycle in a dish. This culture system will provide a platform for elucidating the molecular mechanisms underlying totipotency and the production of oocytes of other mammalian species in culture..
28. Shirane K, Kurimoto K, Yabuta Y, Yamaji M, Satoh J, Ito S, Watanabe A, Hayashi K, Saitou M, Sasaki H., Global Landscape and Regulatory Principles of DNA Methylation Reprogramming for Germ Cell Specification by Mouse Pluripotent Stem Cells., Dev Cell, doi: 10.1016/j.devcel.2016.08.008., 2016.10, Specification of primordial germ cells (PGCs) activates epigenetic reprogramming for totipotency, the elucidation of which remains a fundamental challenge. Here, we uncover regulatory principles for DNA methylation reprogramming during in vitro PGC specification, in which mouse embryonic stem cells (ESCs) are induced into epiblast-like cells (EpiLCs) and then PGC-like cells (PGCLCs). While ESCs reorganize their methylome to form EpiLCs, PGCLCs essentially dilute the EpiLC methylome at constant, yet different, rates between unique sequence regions and repeats. ESCs form hypomethylated domains around pluripotency regulators for their activation, whereas PGCLCs create demethylation-sensitive domains around developmental regulators by accumulating abundant H3K27me3 for their repression. Loss of PRDM14 globally upregulates methylation and diminishes the hypomethylated domains, but it preserves demethylation-sensitive domains. Notably, female ESCs form hypomethylated lamina-associated domains, while female PGCLCs effectively reverse such states into a more normal configuration. Our findings illuminate the unique orchestration of DNA methylation and histone modification reprogramming during PGC specification..
29. Kenjiro Shirane, Kazuki Kurimoto, Yukihiro Yabuta, Masashi Yamaji, Junko Satoh, Shinji Ito, Akira Watanabe, Katsuhiko Hayashi, Mitinori Saitou, Hiroyuki Sasaki, Global Landscape and Regulatory Principles of DNA Methylation Reprogramming for Germ Cell Specification by Mouse Pluripotent Stem Cells, Developmental Cell, 10.1016/j.devcel.2016.08.008, 39, 1, 87-103, 2016.10, Specification of primordial germ cells (PGCs) activates epigenetic reprogramming for totipotency, the elucidation of which remains a fundamental challenge. Here, we uncover regulatory principles for DNA methylation reprogramming during in vitro PGC specification, in which mouse embryonic stem cells (ESCs) are induced into epiblast-like cells (EpiLCs) and then PGC-like cells (PGCLCs). While ESCs reorganize their methylome to form EpiLCs, PGCLCs essentially dilute the EpiLC methylome at constant, yet different, rates between unique sequence regions and repeats. ESCs form hypomethylated domains around pluripotency regulators for their activation, whereas PGCLCs create demethylation-sensitive domains around developmental regulators by accumulating abundant H3K27me3 for their repression. Loss of PRDM14 globally upregulates methylation and diminishes the hypomethylated domains, but it preserves demethylation-sensitive domains. Notably, female ESCs form hypomethylated lamina-associated domains, while female PGCLCs effectively reverse such states into a more normal configuration. Our findings illuminate the unique orchestration of DNA methylation and histone modification reprogramming during PGC specification..
30. Morohaku K, Tanimoto R, Sasaki K, Kawahara-Miki R, Kono T, Hayashi K, Hirao Y, Obata Y., Complete in vitro generation of fertile oocytes from mouse primordial germ cells., Proc Natl Acad Sci U S A, doi: 10.1073/pnas.1603817113., 2016.08, Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogen-receptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells..
31. Kanako Morohaku, Ren Tanimoto, Keisuke Sasaki, Ryouka Kawahara-Miki, Tomohiro Kono, Katsuhiko Hayashi, Yuji Hirao, Yayoi Obata, Complete in vitro generation of fertile oocytes from mouse primordial germ cells, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.1603817113, 113, 32, 9021-9026, 2016.08, Reconstituting gametogenesis in vitro is a key goal for reproductive biology and regenerative medicine. Successful in vitro reconstitution of primordial germ cells and spermatogenesis has recently had a significant effect in the field. However, recapitulation of oogenesis in vitro remains unachieved. Here we demonstrate the first reconstitution, to our knowledge, of the entire process of mammalian oogenesis in vitro from primordial germ cells, using an estrogenreceptor antagonist that promotes normal follicle formation, which in turn is crucial for supporting oocyte growth. The fundamental events in oogenesis (i.e., meiosis, oocyte growth, and genomic imprinting) were reproduced in the culture system. The most rigorous evidence of the recapitulation of oogenesis was the birth of fertile offspring, with a maximum of seven pups obtained from a cultured gonad. Moreover, cryopreserved gonads yielded functional oocytes and offspring in this culture system. Thus, our in vitro system will enable both innovative approaches for a deeper understanding of oogenesis and a new avenue to create and preserve female germ cells..
32. Joseph Saragusty, Sebastian Diecke, Micha Drukker, Barbara Durrant, Inbar Friedrich Ben-Nun, Cesare Galli, Frank Göritz, Katsuhiko Hayashi, Robert Hermes, Susanne Holtze, Stacey Johnson, Giovanna Lazzari, Pasqualino Loi, Jeanne F. Loring, Keisuke Okita, Marilyn B. Renfree, Steven Seet, Thomas Voracek, Jan Stejskal, Oliver A. Ryder, Thomas B. Hildebrandt, Rewinding the process of mammalian extinction, Zoo biology, 10.1002/zoo.21284, 35, 4, 280-292, 2016.07, With only three living individuals left on this planet, the northern white rhinoceros (Ceratotherium simum cottoni) could be considered doomed for extinction. It might still be possible, however, to rescue the (sub)species by combining novel stem cell and assisted reproductive technologies. To discuss the various practical options available to us, we convened a multidisciplinary meeting under the name "Conservation by Cellular Technologies." The outcome of this meeting and the proposed road map that, if successfully implemented, would ultimately lead to a self-sustaining population of an extremely endangered species are outlined here. The ideas discussed here, while centered on the northern white rhinoceros, are equally applicable, after proper adjustments, to other mammals on the brink of extinction. Through implementation of these ideas we hope to establish the foundation for reversal of some of the effects of what has been termed the sixth mass extinction event in the history of Earth, and the first anthropogenic one. Zoo Biol. 35:280-292, 2016..
33. Fereshteh Esfandiari, Omid Mashinchian, Mohammad Kazemi Ashtiani, Mohammad Hossein Ghanian, Katsuhiko Hayashi, Amir Ata Saei, Morteza Mahmoudi, Hossein Baharvand, Possibilities in Germ Cell Research
An Engineering Insight, Trends in biotechnology, 10.1016/j.tibtech.2015.09.004, 33, 12, 735-746, 2015.12, Germ cells (GCs) are responsible for fertility and disruptions in their development or function cause infertility. However, current knowledge about the diverse mechanisms involved in GC development and function is still in its infancy. This is mainly because there are low numbers of GCs, especially during embryonic development. A deeper understanding of GCs would enhance our ability to produce them from stem cells. In addition, such information would enable the production of healthy gametes for infertile couples. In this regard, pluripotent stem cells (PSCs) demonstrated a promising ability to produce GCs in vitro. In this review, we highlight recent advances in the field of tissue engineering that suggest novel strategies to enhance GC research. Advanced therapies for infertility require developing systems for the efficient production of germ cells from stem cells in the laboratory.We discuss recent advances and challenges of germ cell research.Here, we propose engineering approaches for designing an artificial niche for germ cell development in vitro.Novel platforms are demonstrated for the epigenetic analysis of germ cells.Innovative approaches are detailed for the efficient production of germ cells from pluripotent stem cells..
34. Kurimoto K, Yabuta Y, Hayashi K, Ohta H, Kiyonari H, Mitani T, Moritoki Y, Kohri K, Kimura H, Yamamoto T, Katou Y, Shirahige K, Saitou M., Quantitative Dynamics of Chromatin Remodeling during Germ Cell Specification from Mouse Embryonic Stem Cells., Cell Stem Cell, doi: 10.1016/j.stem.2015.03.002., 2015.05, Germ cell specification is accompanied by epigenetic remodeling, the scale and specificity of which are unclear. Here, we quantitatively delineate chromatin dynamics during induction of mouse embryonic stem cells (ESCs) to epiblast-like cells (EpiLCs) and from there into primordial germ cell-like cells (PGCLCs), revealing large-scale reorganization of chromatin signatures including H3K27me3 and H3K9me2 patterns. EpiLCs contain abundant bivalent gene promoters characterized by low H3K27me3, indicating a state primed for differentiation. PGCLCs initially lose H3K4me3 from many bivalent genes but subsequently regain this mark with concomitant upregulation of H3K27me3, particularly at developmental regulatory genes. PGCLCs progressively lose H3K9me2, including at lamina-associated perinuclear heterochromatin, resulting in changes in nuclear architecture. T recruits H3K27ac to activate BLIMP1 and early mesodermal programs during PGCLC specification, which is followed by BLIMP1-mediated repression of a broad range of targets, possibly through recruitment and spreading of H3K27me3. These findings provide a foundation for reconstructing regulatory networks of the germline epigenome..
35. Kazuki Kurimoto, Yukihiro Yabuta, Katsuhiko Hayashi, Hiroshi Ohta, Hiroshi Kiyonari, Tadahiro Mitani, Yoshinobu Moritoki, Kenjiro Kohri, Hiroshi Kimura, Takuya Yamamoto, Yuki Katou, Katsuhiko Shirahige, Mitinori Saitou, Quantitative dynamics of chromatin remodeling during germ cell specification from mouse embryonic stem cells, Cell stem cell, 10.1016/j.stem.2015.03.002, 16, 5, 517-532, 2015.05, Germ cell specification is accompanied by epigenetic remodeling, the scale and specificity of which are unclear. Here, we quantitatively delineate chromatin dynamics during induction of mouse embryonic stem cells (ESCs) to epiblast-like cells (EpiLCs) and from there into primordial germ cell-like cells (PGCLCs), revealing large-scale reorganization of chromatin signatures including H3K27me3 and H3K9me2 patterns. EpiLCs contain abundant bivalent gene promoters characterized by low H3K27me3, indicating a state primed for differentiation. PGCLCs initially lose H3K4me3 from many bivalent genes but subsequently regain this mark with concomitant upregulation of H3K27me3, particularly at developmental regulatory genes. PGCLCs progressively lose H3K9me2, including at lamina-associated perinuclear heterochromatin, resulting in changes in nuclear architecture. T recruits H3K27ac to activate BLIMP1 and early mesodermal programs during PGCLC specification, which is followed by BLIMP1-mediated repression of a broad range of targets, possibly through recruitment and spreading of H3K27me3. These findings provide a foundation for reconstructing regulatory networks of the germline epigenome..
36. Kimura T, Kaga Y, Ohta H, Odamoto M, Sekita Y, Li K, Yamano N, Fujikawa K, Isotani A, Sasaki N, Toyoda M, Hayashi K, Okabe M, Shinohara T, Saitou M, Nakano T., Induction of Primordial Germ Cell-Like Cells from Mouse Embryonic Stem Cells by ERK Signal Inhibition., Stem Cells, doi: 10.1002/stem.1781., 2014.10, Primordial germ cells (PGCs) are embryonic germ cell precursors. Specification of PGCs occurs under the influence of mesodermal induction signaling during in vivo gastrulation. Although bone morphogenetic proteins and Wnt signaling play pivotal roles in both mesodermal and PGC specification, the signal regulating PGC specification remains unknown. Coculture of mouse embryonic stem cells (ESCs) with OP9 feeder cells induces mesodermal differentiation in vitro. Using this mesodermal differentiation system, we demonstrated that PGC-like cells were efficiently induced from mouse ESCs by extracellular signal-regulated kinase (ERK) signaling inhibition. Inhibition of ERK signaling by a MAPK/ERK kinase (MEK) inhibitor upregulated germ cell marker genes but downregulated mesodermal genes. In addition, the PGC-like cells showed downregulation of DNA methylation and formed pluripotent stem cell colonies upon treatment with retinoic acid. These results show that inhibition of ERK signaling suppresses mesodermal differentiation but activates germline differentiation program in this mesodermal differentiation system. Our findings provide a new insight into the signaling networks regulating PGC specification..
37. Katsuhiko Hayashi, Mitinori Saitou, Perspectives of germ cell development in vitro in mammals, Animal Science Journal, 10.1111/asj.12199, 85, 6, 617-626, 2014.06, Pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are able to differentiate into all cell lineages of the embryo proper, including germ cells. This pluripotent property has a huge impact on the fields of regenerative medicine, developmental biology and reproductive engineering. Establishing the germ cell lineage from ESCs/iPSCs is the key biological subject, since it would contribute not only to dissection of the biological processes of germ cell development but also to production of unlimited numbers of functional gametes in vitro. Toward this goal, we recently established a culture system that induces functional mouse primordial germ cells (PGCs), precursors of all germ cells, from mouse ESCs/iPSCs. The successful in vitro production of PGCs arose from the study of pluripotent cell state, the signals inducing PGCs and the technology of transplantation. However, there are many obstacles to be overcome for the robust generation of mature gametes or for application of the culture system to other species, including humans and livestock. In this review, we discuss the requirements for a culture system to generate the germ cell lineage from ESCs/iPSCs..
38. Katsuhiko Hayashi, Mitinori Saitou, Generation of oocytes from mouse ES/iPS cells, Journal of Mammalian Ova Research, 10.1274/jmor.31.70, 31, 3, 70-78, 2014.01, One of the paramount goals in reproductive biology is to produce functional oocytes in culture through a series of differentiation processes that accurately mimic those in vivo. Such a culture system would provide a larger number of oocytes than those available in vivo, which would be of help in the elucidation of mechanisms underlying germ cell development. All germ cells originate from a small group of primordial germ cells (PGCs) that segregate from somatic cell lineages at an early developmental stage. Recently, we developed a culture system in which embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) differentiate into PGC-like cells (PGCLCs) through an epiblast-like cell population. PGCLCs are capable of differentiating into functional oocytes when transplanted into the ovarian bursa with E12.5 gonadal somatic cells. In this review, we introduce our differentiation method and discuss possible applications of this culture system..
39. Tohru Kimura, Yoshiaki Kaga, Hiroshi Ohta, Mika Odamoto, Yoichi Sekita, Kunpeng Li, Noriko Yamano, Keita Fujikawa, Ayako Isotani, Norihiko Sasaki, Masashi Toyoda, Katsuhiko Hayashi, Masaru Okabe, Takashi Shinohara, Mitinori Saitou, Toru Nakano, Induction of primordial germ cell-like cells from mouse embryonic stem cells by ERK signal inhibition, STEM CELLS, 10.1002/stem.1781, 32, 10, 2668-2678, 2014, Primordial germ cells (PGCs) are embryonic germ cell precursors. Specification of PGCs occurs under the influence of mesodermal induction signaling during in vivo gastrulation. Although bone morphogenetic proteins and Wnt signaling play pivotal roles in both mesodermal and PGC specification, the signal regulating PGC specification remains unknown. Coculture of mouse embryonic stem cells (ESCs) with OP9 feeder cells induces mesodermal differentiation in vitro. Using this mesodermal differentiation system, we demonstrated that PGC-like cells were efficiently induced from mouse ESCs by extracellular signal-regulated kinase (ERK) signaling inhibition. Inhibition of ERK signaling by a MAPK/ERK kinase (MEK) inhibitor upregulated germ cell marker genes but downregulated mesodermal genes. In addition, the PGC-like cells showed downregulation of DNA methylation and formed pluripotent stem cell colonies upon treatment with retinoic acid. These results show that inhibition of ERK signaling suppresses mesodermal differentiation but activates germline differentiation program in this mesodermal differentiation system. Our findings provide a new insight into the signaling networks regulating PGC specification..
40. Shinya Aramaki, Katsuhiko Hayashi, Kazuki Kurimoto, Hiroshi Ohta, Yukihiro Yabuta, Hiroko Iwanari, Yasuhiro Mochizuki, Takao Hamakubo, Yuki Kato, Katsuhiko Shirahige, Mitinori Saitou, A mesodermal factor, T, specifies mouse germ cell fate by directly activating germline determinants, Developmental Cell, 10.1016/j.devcel.2013.11.001, 27, 5, 516-529, 2013.12, Germ cells ensure reproduction and heredity. In mice, primordial germ cells (PGCs), the precursors for spermatozoa and oocytes, are induced in pluripotent epiblast by BMP4 and WNT3, yet the underlying mechanism remains unclear. Here, using an invitro PGC specification system, we show that WNT3 induces many transcription factors associated withmesoderm in epiblast-like cells through β-CATENIN. Among these, T (BRACHYURY), a classical and conserved mesodermal factor, was essential for robust activation of Blimp1 and Prdm14, two of the germline determinants. T, but not SMAD1 or TCF1, binds distinct regulatory elements of both Blimp1 and Prdm14 and directly upregulates these genes, delineating the downstream PGC program. Without BMP4, a program induced by WNT3 prevents T from activating Blimp1 and Prdm14, demonstrating a permissive role of BMP4 in PGC specification. These findings establish the key signaling mechanism for, and a fundamental role of a mesodermal factor in, mammalian PGC specification..
41. Ryushin Mizuta, Shinsuke Araki, Makoto Furukawa, Yuki Furukawa, Syota Ebara, Daisuke Shiokawa, Katsuhiko Hayashi, Sei Ichi Tanuma, Daisuke Kitamura, DNase γ is the effector endonuclease for internucleosomal DNA fragmentation in necrosis, PloS one, 10.1371/journal.pone.0080223, 8, 12, 2013.12, Apoptosis and necrosis, two major forms of cell death, can be distinguished morphologically and biochemically. Internucleosomal DNA fragmentation (INDF) is a biochemical hallmark of apoptosis, and caspase-activated DNase (CAD), also known as DNA fragmentation factor 40 kDa (DFF40), is one of the major effector endonucleases. DNase γ, a Mg2+/Ca2+-dependent endonuclease, is also known to generate INDF but its role among other apoptosis-associated endonucleases in cell death is unclear. Here we show that (i) INDF occurs even during necrosis in cell lines, primary cells, and in tissues of mice in vivo, and (ii) DNase c, but not CAD, is the effector endonuclease for INDF in cells undergoing necrosis. These results document a previously unappreciated role for INDF in necrosis and define its molecular basis. Copyright:.
42. Bernhard Payer, Michael Rosenberg, Masashi Yamaji, Yukihiro Yabuta, Michiyo Koyanagi-Aoi, Katsuhiko Hayashi, Shinya Yamanaka, Mitinori Saitou, Jeannie T. Lee, Tsix RNA and the germline factor, PRDM14, link X reactivation and stem cell reprogramming, Molecular Cell, 10.1016/j.molcel.2013.10.023, 52, 6, 805-818, 2013.12, Transitions between pluripotent and differentiated states are marked by dramatic epigenetic changes. Cellular differentiation is tightly linked to X chromosome inactivation (XCI), whereas reprogramming to induced pluripotent stem cells (iPSCs) is associated with X chromosome reactivation (XCR). XCR reverses the silent state of the inactive X, occurring in mouse blastocysts and germ cells. In spite of its importance, little is known about underlying mechanisms. Here, we examine the role of the long noncoding Tsix RNA and the germline factor, PRDM14. In blastocysts, XCR is perturbed by mutation of either Tsix or Prdm14. In iPSCs, XCR is disrupted only by PRDM14 deficiency, which also affects iPSC derivation and maintenance. We show that Tsix and PRDM14 directly link XCR to pluripotency: first, PRDM14 represses Rnf12 by recruiting polycomb repressive complex 2; second, Tsix enables PRDM14 to bind Xist. Thus, our study provides functional and mechanistic links between cellular and X chromosome reprogramming..
43. Katsuhiko Hayashi, Mitinori Saitou, Generation of eggs from mouse embryonic stem cells and induced pluripotent stem cells, Nat Protoc. , 10.1038/nprot.2013.090, 8, 8, 1513-1524, 2013.08, Oogenesis is an integrated process through which an egg acquires the potential for totipotency, a fundamental condition for creating new individuals. Reconstitution of oogenesis in a culture that generates eggs with proper function from pluripotent stem cells (PSCs) is therefore one of the key goals in basic biology as well as in reproductive medicine. Here we describe a stepwise protocol for the generation of eggs from mouse PSCs, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). ESCs and iPSCs are first induced into primordial germ cell-like cells (PGCLCs) that are in turn aggregated with somatic cells of female embryonic gonads, the precursors for adult ovaries. Induction of PGCLCs followed by aggregation with the somatic cells takes up to 8 d. The aggregations are then transplanted under the ovarian bursa, in which PGCLCs grow into germinal vesicle (GV) oocytes in similar to 1 month. The PGCLC-derived GV oocytes can be matured into eggs in 1 d by in vitro maturation (IVM), and they can be fertilized with spermatozoa by in vitro fertilization (IVF) to obtain healthy and fertile offspring. This method provides an initial step toward reconstitution of the entire process of oogenesis in vitro..
44. Fumio Nakaki, Katsuhiko Hayashi, Hiroshi Ohta, Kazuki Kurimoto, Yukihiro Yabuta, Mitinori Saitou, Induction of mouse germ-cell fate by transcription factors in vitro, Nature, 10.1038/nature12417, 501, 7466, 222-226, 2013.08, The germ-cell lineage ensures the continuity of life through the generation of male and female gametes, which unite to form a totipotent zygote. We have previously demonstrated that, by using cytokines, embryonic stem cells and induced pluripotent stem cells can be induced into epiblast-like cells (EpiLCs) and then into primordial germ cell (PGC)-like cells with the capacity for both spermatogenesis and oogenesis, creating an opportunity for understanding and regulating mammalian germ-cell development in both sexes in vitro. Here we show that, without cytokines, simultaneous overexpression of three transcription factors, Blimp1 (also known as Prdm1), Prdm14 and Tfap2c (also known as AP2γ), directs EpiLCs, but not embryonic stem cells, swiftly and efficiently into a PGC state. Notably, Prdm14 alone, but not Blimp1 or Tfap2c, suffices for the induction of the PGC state in EpiLCs. The transcription-factor- induced PGC state, irrespective of the transcription factors used, reconstitutes key transcriptome and epigenetic reprogramming in PGCs, but bypasses a mesodermal program that accompanies PGC or PGC-like-cell specification by cytokines including bone morphogenetic protein 4. Notably, the transcription-factor-induced PGC-like cells contribute to spermatogenesis and fertile offspring. Our findings provide a new insight into the transcriptional logic for PGC specification, and create a foundation for the transcription-factor-based reconstitution and regulation of mammalian gametogenesis..
45. Masashi Yamaji, Jun Ueda, Katsuhiko Hayashi, Hiroshi Ohta, Yukihiro Yabuta, Kazuki Kurimoto, Ryuichiro Nakato, Yasuhiro Yamada, Katsuhiko Shirahige, Mitinori Saitou, PRDM14 ensures naive pluripotency through dual regulation of signaling and epigenetic pathways in mouse embryonic stem cells, Cell stem cell, 10.1016/j.stem.2012.12.012, 12, 3, 368-382, 2013.03, In serum, mouse embryonic stem cells (mESCs) fluctuate between a naive inner cell mass (ICM)-like state and a primed epiblast-like state, but when cultured with inhibitors of the mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 pathways (2i), they are harnessed exclusively in a distinct naive pluropotent state, the ground state, that more faithfully recapitulates the ICM. Understanding the mechanism underlying this naive pluripotent state will be critical for realizing the full potential of ESCs. We show here that PRDM14, a PR-domain-containing transcriptional regulator, ensures naive pluripotency through a dual mechanism: antagonizing activation of the fibroblast growth factor receptor (FGFR) signaling by the core pluripotency transcriptional circuitry, and repressing expression of de novo DNA methyltransferases that modify the epigenome to a primed epiblast-like state. PRDM14 exerts these effects by recruiting polycomb repressive complex 2 (PRC2) specifically to key targets and repressing their expression..
46. Katsuhiko Hayashi, Mitinori Saitou, Stepwise differentiation from Naïve state pluripotent stem cells to functional primordial germ cells through an epiblast-like state, Epiblast Stem Cells Methods and Protocols, 10.1007/978-1-62703-628-3_13, 175-183, 2013, A group of pluripotent cells appearing during mammalian embryogenesis is the source for all the cell lineages that compose the embryo proper. In mice, pluripotent cells are first established in the inner cell mass (ICM) of the preimplantation blastocyst. After implantation, the ICM soon transforms into a cup-shaped epithelium, called the postimplantation epiblast. The two types of pluripotent cells, the ICM and postimplantation epiblast cells, are distinct, based on the differences in their gene expression profiles, epigenetic status, and differentiation capacity. During gastrulation, some of the postimplantation epiblast cells adjacent to the extraembryonic ectoderm are specified as primordial germ cells (PGCs), precursors of the germ cell lineage, in response to bone morphogenetic protein 4 (BMP4). Recently, we succeeded in reconstituting epiblast differentiation and PGC specification in vitro using pluripotent stem cells. Here, we describe the culture method of a stepwise differentiation from pluripotent stem cells to functional PGCs..
47. Katsuhiko Hayashi, Sugako Ogushi, Kazuki Kurimoto, So Shimamoto, Hiroshi Ohta, Mitinori Saitou, Offspring from Oocytes Derived from in Vitro Primordial Germ Cell-like Cells in Mice, Science, 10.1126/science.1226889, 338, 6109, 971-975, 2012.10, Reconstitution of female germ cell development in vitro is a key challenge in reproductive biology and medicine. We show here that female (XX) embryonic stem cells and induced pluripotent stem cells in mice are induced into primordial germ cell-like cells (PGCLCs), which, when aggregated with female gonadal somatic cells as reconstituted ovaries, undergo X-reactivation, imprint erasure, and cyst formation, and exhibit meiotic potential. Upon transplantation under mouse ovarian bursa, PGCLCs in the reconstituted ovaries mature into germinal vesicle-stage oocytes, which then contribute to fertile offspring after in vitro maturation and fertilization. Our culture system serves as a robust foundation for the investigation of key properties of female germ cells, including the acquisition of totipotency, and for the reconstitution of whole female germ cell development in vitro..
48. Katsuhiko Hayashi, Mitinori Saitou, Generation of functional primordial germ cells from pluripotent stem cells, Journal of Mammalian Ova Research, 10.1274/jmor.29.2, 29, 1, 2-10, 2012.06, Primordial germ cells (PGCs), origin of the germ cell lineage, arise from epiblast cells in response to BMP4 secreted by adjacent extraembryonic ectoderm. We recently reconstituted the PGC specification in vitro using mouse embryonic stem cells (mESCs) as well as induced pluripotent stem cells (iPSCs). In the culture system, mESCs/iPSCs first differentiated into epiblastlike cells (EpiLCs) and then induced PGC-like cells from the EpiLCs. This manner of differentiation from mESCs to PGCs reproduces the manner of PGC specification in vivo. PGCs produced from mESCs, termed PGC-like cells (PGCLCs), were fully potent, since they differentiated into spermatozoa and in turn the fertilized eggs with the spermatozoa gave rise to healthy individuals. Although many attempts have been made to produce fully potent PGCs, this study was the first study demonstrating the successful production of healthy individuals from PGCLCs. This achievement was made possible by knowledge accumulated on the manner of PGC specification in vivo, the nature of self-renewing pluripotent stem cells, and growth factors endowing EpiLC formation and PGCLC induction in vitro. This article reviews the research advance that made it possible to reconstitute PGC specification in vitro from mESCs..
49. Astrid Gillich, Siqin Bao, Nils Grabole, Katsuhiko Hayashi, Matthew W.B. Trotter, Vincent Pasque, Erna Magnúsdóttir, M. Azim Surani, Epiblast stem cell-based system reveals reprogramming synergy of germline factors, Cell stem cell, 10.1016/j.stem.2012.01.020, 10, 4, 425-439, 2012.04, Epigenetic reprogramming in early germ cells is critical toward the establishment of totipotency, but investigations of the germline events are intractable. An objective cell culture-based system could provide mechanistic insight on how the key determinants of primordial germ cells (PGCs), including Prdm14, induce reprogramming in germ cells to an epigenetic ground state. Here we show a Prdm14-Klf2 synergistic effect that can accelerate and enhance reversion of mouse epiblast stem cells (epiSCs) to a naive pluripotent state, including X reactivation and DNA demethylation. Notably, Prdm14 alone has little effect on epiSC reversion, but it enhances the competence for reprogramming and potentially PGC specification. Reprogramming of epiSCs by the combinatorial effect of Prdm14-Klf2 involves key epigenetic changes, which might have an analogous role in PGCs. Our study provides a paradigm toward a systematic analysis of how other key genes contribute to complex and dynamic events of reprogramming in the germline..
50. Jamie Trott, Katsuhiko Hayashi, Azim Surani, M. Madan Babu, Alfonso Martinez-Arias, Dissecting ensemble networks in ES cell populations reveals micro-heterogeneity underlying pluripotency, Molecular BioSystems, 10.1039/c1mb05398a, 8, 3, 744-752, 2012.03, Analysis of transcription at the level of single cells in prokaryotes and eukaryotes has revealed the existence of heterogeneities in the expression of individual genes within genetically homogeneous populations. This variation is an emerging hallmark of populations of Embryonic Stem (ES) cells and has been ascribed to the stochasticity associated with the biochemical events that mediate gene expression. It has been suggested that these heterogeneities play a role in the maintenance of pluripotency. However, for the most part, studies have focused on individual genes in large cell populations. Here we use an existing dataset on the expression of eight genes involved in pluripotency in eighty-three ES cells to create Gene Regulatory Networks (GRNs) at the single cell level. We observe widespread heterogeneities in the expression of the eight genes, but analysis of correlations within individual cells reveals three distinct classes centered on the expression of Nanog, a marker of pluripotency, and Fgf5, a gene associated with differentiation: high levels of Nanog and low levels of Fgf5, low levels of Nanog and high levels of Fgf5, and low levels of both. Each of these classes is associated with a collection of active sub-networks, with differing degrees of connectivity between their elements, which define a cellular state: self-renewal, primed for differentiation or transition between the two. Though every cell should be governed by the same network, the active sub-networks may emerge due to considerations such as variation in (i) the expression level of active transcription factors (e.g. through post-translational modification or ligand/co-factor availability) or (ii) access to the target gene locus (e.g. via changes in chromatin status or epigenetic modifications). We conclude that heterogeneities in gene expression should not be interpreted as representing different states of a single unique network, but as a reflection of the activity of different sub-networks in sub-populations of cells..
51. Katsuhiko Hayashi, Hiroshi Ohta, Kazuki Kurimoto, Shinya Aramaki, Mitinori Saitou, Reconstitution of the Mouse Germ Cell Specification Pathway in Culture by Pluripotent Stem Cells, 10.1016/j.cell.2011.06.052, 146, 4 , 519-532 , 2011.08, The generation of properly functioning gametes in vitro requires reconstitution of the multistepped pathway of germ cell development. We demonstrate here the generation of primordial germ cell-like cells (PGCLCs) in mice with robust capacity for spermatogenesis. PGCLCs were generated from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) through epiblast-like cells (EpiLCs), a cellular state highly similar to pregastrulating epiblasts but distinct from epiblast stem cells (EpiSCs). Reflecting epiblast development, EpiLC induction from ESCs/iPSCs is a progressive process, and EpiLCs highly competent for the PGC fate are a transient entity. The global transcription profiles, epigenetic reprogramming, and cellular dynamics during PGCLC induction from EpiLCs meticulously capture those associated with PGC specification from the epiblasts. Furthermore, we identify Integrin-beta 3 and SSEA1 as markers that allow the isolation of PGCLCs with spermatogenic capacity from tumorigenic undifferentiated cells. Our findings provide a paradigm for the first step of in vitro gametogenesis..
52. Astrid Gillich, Katsuhiko Hayashi, Switching stem cell state through programmed germ cell reprogramming, Differentiation, 10.1016/j.diff.2011.01.003, 81, 5, 281-291, 2011.06, Depending on their origin, embryo-derived stem cells have distinct properties that largely correspond to their counterpart in vivo. Mouse epiblast stem cells derived from post-implantation embryos differ from embryonic stem cells derived from blastocysts in their transcriptional and epigenetic profile, their morphology and culture requirements. When maintained in appropriate conditions, the cells keep self-renewing and do not adopt a different state. Recent studies, however, show that it is possible to convert between stem cell states. Here we review recent advances to induce stem cell state changes and we consider the potential of germ cell-mediated reprogramming for the conversion. Since the properties of mouse epiblast stem cells are similar to human embryonic stem cells, we discuss the significance of stem cell conversion and germ cell-mediated reprogramming in humans..
53. Sophie A. Hanina, William Mifsud, Thomas A. Down, Katsuhiko Hayashi, Dónal O'Carroll, Kaiqin Lao, Eric A. Miska, M. Azim Surani, Genome-wide identification of targets and function of individual MicroRNAs in mouse embryonic stem cells., PLoS Genetics, 6, 10, 2010.12, Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes and upregulating pluripotency-associated genes such as Lin28..
54. Sophie A. Hanina, William Mifsud, Thomas A. Down, Katsuhiko Hayashi, Dónal O'Carroll, Kaiqin Lao, Eric A. Miska, M. Azim Surani, Genome-wide identification of targets and function of individual microRNAs in mouse embryonic stem cells, PLoS genetics, 10.1371/journal.pgen.1001163, 6, 10, 1-13, 2010.10, Mouse Embryonic Stem (ES) cells express a unique set of microRNAs (miRNAs), the miR-290-295 cluster. To elucidate the role of these miRNAs and how they integrate into the ES cell regulatory network requires identification of their direct regulatory targets. The difficulty, however, arises from the limited complementarity of metazoan miRNAs to their targets, with the interaction requiring as few as six nucleotides of the miRNA seed sequence. To identify miR-294 targets, we used Dicer1-null ES cells, which lack all endogenous mature miRNAs, and introduced just miR-294 into these ES cells. We then employed two approaches to discover miR-294 targets in mouse ES cells: transcriptome profiling using microarrays and a biochemical approach to isolate mRNA targets associated with the Argonaute2 (Ago2) protein of the RISC (RNA Induced Silencing Complex) effector, followed by RNA-sequencing. In the absence of Dicer1, the RISC complexes are largely devoid of mature miRNAs and should therefore contain only transfected miR-294 and its base-paired targets. Our data suggest that miR-294 may promote pluripotency by regulating a subset of c-Myc target genes and upregulating pluripotency-associated genes such as Lin28..
55. Mylah Villacorte, Kentaro Suzuki, Katsuhiko Hayashi, Susana Chuva de Sousa Lopes, Ryuma Haraguchi, Makoto M. Taketo, Naomi Nakagata, Gen Yamada, Antagonistic crosstalk of Wnt/β-catenin/Bmp signaling within the Apical Ectodermal Ridge (AER) regulates interdigit formation, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2009.12.109, 391, 4, 1653-1657, 2010.01, Digit and interdigit (D/ID) development is one of the important research fields in molecular developmental biology. Interdigital cell death (ICD) is a morphogenetic event which has been considered as an essential process for D/ID formation. Although some growth factors including Bmp and Fgf signaling can modulate ICD, growth factor crosstalk regulating ICD is poorly understood. Wnt canonical pathway and Bmp signal crosstalk has been considered as the essential growth factor crosstalk in organogenesis. To elucidate the crosstalk to regulate the D/ID formation, we analyzed conditional mutant mice with limb bud ectoderm expressing constitutively activated β-catenin signaling. We showed that modulation of Wnt/β-catenin signal in the limb ectoderm including the AER regulates ID apoptosis. We also demonstrated that Wnt/β-catenin signaling in the ectoderm can positively regulate Fgf8 possibly antagonizing the epithelial derived Bmp signaling. Human birth defects for digit abnormalities have been known to be affected by multiple parameters. Elucidation of the potential mechanisms underlying such D/ID development is an urgent medical issue to be solved. This work would be one of the first studies showing essential growth factor cascades in the D/ID formation..
56. Katsuhiko Hayashi, Azim Surani, Self-renewing epiblast stem cells exhibit continual delineation of germ cells with epigenetic reprogramming in vitro, 10.1242/dev.037747, 136, 21, 3549-3556, 2009.11, Pluripotent epiblast stem cells (EpiSCs) derived from postimplantation embryos exhibit properties that are characteristically different when compared with pluripotent embryonic stem cells (ESCs) derived from mouse blastocysts. However, EpiSCs are relatively less well characterised compared with ESCs. In particular, the relationship between EpiSCs and primordial germ cells (PGCs) is unknown, and is worthy of investigation because PGCs originate from postimplantation epiblast cells in vivo. We show that EpiSCs have an infinite capacity for generating PGCs, under conditions that sustain their pluripotency and self-renewal. These PGCs generated in vitro show appropriate transcriptional and epigenetic reprogramming events and are able to develop further into late germ cells. Notably, the PGCs can, in turn, be induced to undergo dedifferentiation into pluripotent embryonic germ cells (EGCs), which resemble ESCs and not the EpiSC from which they are derived. Our observations demonstrate intrinsic reprogramming during specification of PGCs that results in the erasure of epigenetic memory of EpiSCs following reactivation of the X-chromosome, DNA demethylation and re-expression of key pluripotency genes. This study provides novel insights into the nature and properties of EpiSCs, and introduces an in vitro model system that will be useful for investigations on PGC specification and on mechanisms regulating epigenetic reprogramming in germ cells..
57. Siqin Bao, Fuchou Tang, Xihe Li, Katsuhiko Hayashi, Astrid Gillich, Kaiqin Lao, M. Azim Surani, Epigenetic reversion of post-implantation epiblast to pluripotent embryonic stem cells, Nature, 10.1038/nature08534, 461, 7268, 1292-1295, 2009.10, The pluripotent state, which is first established in the primitive ectoderm cells of blastocysts, is lost progressively and irreversibly during subsequent development. For example, development of post-implantation epiblast cells from primitive ectoderm involves significant transcriptional and epigenetic changes, including DNA methylation and X chromosome inactivation, which create a robust epigenetic barrier and prevent their reversion to a primitive-ectoderm-like state. Epiblast cells are refractory to leukaemia inhibitory factor (LIF)-STAT3 signalling, but they respond to activin/basic fibroblast growth factor to form self-renewing epiblast stem cells (EpiSCs), which exhibit essential properties of epiblast cells and that differ from embryonic stem (ES) cells derived from primitive ectoderm. Here we show reprogramming of advanced epiblast cells from embryonic day 5.5-7.5 mouse embryos with uniform expression of N-cadherin and inactive X chromosome to ES-cell-like cells (rESCs) in response to LIF-STAT3 signalling. Cultured epiblast cells overcome the epigenetic barrier progressively as they proceed with the erasure of key properties of epiblast cells, resulting in DNA demethylation, X reactivation and expression of E-cadherin. The accompanying changes in the transcriptome result in a loss of phenotypic and epigenetic memory of epiblast cells. Using this approach, we report reversion of established EpiSCs to rESCs. Moreover, unlike epiblast and EpiSCs, rESCs contribute to somatic tissues and germ cells in chimaeras. Further studies may reveal how signalling-induced epigenetic reprogramming may promote reacquisition of pluripotency..
58. Katsuhiko Hayashi, PGC specification in vivo and in vitro, Journal of Mammalian Ova Research, 10.1274/jmor.26.171, 26, 4, 171-177, 2009.10, Primordial germ cells (PGCs), the precursors of the germ cells, arise from pluripotent epiblast cells during gastrulation in the mouse embryo. During early PGC development, there are a series of cellular events, each of which is important in the acquisition of totipotency. Pluripotent stem cells, such as embryonic stem (ES) cells, are a potential source of PGCs in vitro. Accumulating evidence suggests that ES cells can differentiate into PGCs via either embryoid body formation or monolayer cell culture. However, it remains unclear whether these in vitro-derived PGCs properly accomplish all developmental processes during PGC specification in vivo. This article reviews current studies of PGC specification both in vivo and in vitro, shelding light on the significance of PGC specification, and provides a guideline for verification in the process of PGC production in vitro..
59. Katsuhiko Hayashi, M. Azim Surani, Resetting the Epigenome beyond Pluripotency in the Germline, Cell stem cell, 10.1016/j.stem.2009.05.007, 4, 6, 493-498, 2009.06, Germ cells undergo comprehensive epigenetic reprogramming toward acquiring fitness for pluripotency and totipotency. Notably, the full extent of the epigenetic reprogramming experienced by germ cells remains unmatched by attempts to experimentally restore pluripotency in somatic cells. We propose that the defects present in experimentally generated cells are corrected upon differentiation into the germ cell lineage, as has been observed in cases of germline transmission. Unraveling the mechanisms responsible for germ cell-specific epigenetic reprogramming will likely have important implications for both basic and clinical stem cell research..
60. Joji Nakayama, Mutsumi Yamamoto, Katsuhiko Hayashi, Hitoshi Satoh, Kenji Bundo, Masato Kubo, Ryo Goitsuka, Michael A. Farrar, Daisuke Kitamura, BLNK suppresses pre B-cell leukemogenesis through inhibition of JAK3, Blood, 10.1182/blood-2008-07-166355, 113, 7, 1483-1492, 2009.02, Pre - B-cell leukemia spontaneously develops in BLNK-deficient mice, and pre - B-cell acute lymphoblastic leukemia cells in children often lack BLNK protein expression, demonstrating that BLNK functions as a tumor suppressor. However, the mechanism by which BLNK suppresses pre - B-cell leukemia, as well as the identification of other genetic alterations that collaborate with BLNK deficiency to cause leukemogenesis, are still unknown. Here, we demonstrate that the JAK3/STAT5 signaling pathway is constitutively activated in pre-B leukemia cells derived from BLNK -/- mice, mostly due to autocrine production of IL-7. Inhibition of IL-7R signaling or JAK3/STAT5 activity resulted in the induction of p27 kip1 expression and cell-cycle arrest, accompanied by apoptosis in the leukemia cells. Transgene-derived constitutively active STAT5 (STAT5b-CA) strongly synergized with the loss of BLNK to initiate leukemia in vivo. In the leukemia cells, exogenously expressed BLNK inhibited autocrine JAK3/ STAT5 signaling, resulting in p27 kip1 induction, cell-cycle arrest, and apoptosis. BLNK-inhibition of JAK3 was dependent on the binding of BLNK to JAK3. These data indicate that BLNK normally regulates IL-7 - dependent proliferation and survival of pre - B cells through direct inhibition of JAK3. Thus, somatic loss of BLNK and concomitant mutations leading to constitutive activation of Jak/ STAT5 pathway result in the generation of pre - B-cell leukemia..
61. M. A. Surani, G. Durcova-Hills, P. Hajkova, K. Hayashi, W. W. Tee, Germ line, stem cells, and epigenetic reprogramming, Cold Spring Harbor Symposia on Quantitative Biology, 10.1101/sqb.2008.73.015, 73, 9-15, 2008.12, The germ cell lineage has the unique attribute of generating the totipotent state. Development of blastocysts from the totipotent zygote results in the establishment of pluripotent primitive ectoderm cells in the inner cell mass of blastocysts, which subsequently develop into epiblast cells in postimplantation embryos. The germ cell lineage in mice originates from these pluripotent epiblast cells of postimplantation embryos in response to specific signals. Pluripotent stem cells and unipotent germ cells share some fundamental properties despite significant phenotypic differences between them. Additionally, early primordial germ cells can be induced to undergo dedifferentiation into pluripotent embryonic germ cells. Investigations on the relationship between germ cells and pluripotent stem cells may further elucidate the nature of the pluripotent state. Furthermore, comprehensive epigenetic reprogramming of the genome in early germ cells, including extensive erasure of epigenetic modifications, is a critical step toward establishment of totipotency. The mechanisms involved may be relevant for gaining insight into events that lead to reprogramming of somatic cells into pluripotent stem cells..
62. Katsuhiko Hayashi, Susana Lopes, Fuchou Tang, Azim Surani, Dynamic Equilibrium and Heterogeneity of Mouse Pluripotent Stem Cells with Distinct Functional and Epigenetic States, 10.1016/j.stem.2008.07.027, 3, 4, 391-401, 2008.10, Embryonic stem cells (ESCs) are apparently homogeneous self-renewing cells, but we observed heterogeneous expression of Stella in ESCs, which is a marker of pluripotency and germ cells. Here we show that, whereas Stella-positive ESCs were like the inner cell mass (ICM), Stella-negative cells were like the epiblast cells. These states were interchangeable, which reflects the metastability and plasticity of ESCs. The established equilibrium was skewed reversibly in the absence of signals from feeder cells, which caused a marked shift toward an epiblast-like state, while trichostatin A, an inhibitor of histone deactelylase, restored Stella-positive population. The two populations also showed different histone modifications and striking functional differences, as judged by their potential for differentiation. The Stella-negative ESCs were more like the postimplantation epiblast-derived stem cells (EpiSCs), albeit the stella locus was repressed by DNA methylation in the latter, which signifies a robust epigenetic boundary between ESCs and EpiSCs..
63. Fuchou Tang, Katsuhiko Hayashi, Masahiro Kaneda, Kaiqin Lao, M. Azim Surani, A sensitive multiplex assay for piRNA expression, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2008.03.035, 369, 4, 1190-1194, 2008.05, PIWI-interacting RNAs (piRNAs) are a new class of small RNAs specifically expressed in male germ cells. It is known to bind to PIWI class of Argonaute proteins, Mili and Miwi. To help to decipher the mechanism of piRNA function, here, we report a real time PCR-based multiplex assay for piRNA expression. Firstly, we showed that the assay specifically detects piRNA expression in adult testis, consistent with the Northern blot result. The method we developed can simultaneously detect at least eight piRNAs using only 10 pg total RNA, which is equivalent to the RNA present in a single cell. This is five to six order magnitude more sensitive than corresponding Northern blot assays. Finally we used this assay to analyze eight piRNAs expression in mouse primordial germ cells (PGCs) in genital ridges from E12.5, at the time when piRNA-binding protein Mili starts to be detected in PGCs. This multiplex piRNA assay can be further expanded to assay a few hundred of piRNAs simultaneously from as little as total RNA from a single cell. This approach will help to understand the mechanism and function of piRNAs during germ cell development..
64. Masashi Watanabe, Yuri Takagi, Motoko Kotani, Yasushi Hara, Ayako Inamine, Katsuhiko Hayashi, Shuhei Ogawa, Kei Takeda, Kazunari Tanabe, Ryo Abe, Down-regulation of ICOS ligand by interaction with ICOS functions as a regulatory mechanism for immune responses, Journal of Immunology, 10.4049/jimmunol.180.8.5222, 180, 8, 5222-5234, 2008.04, Although it is well-known that the ICOS-ICOS ligand (ICOSL) costimulatory pathway is important for many immune responses, recent accumulated evidence suggests that dysregulation of this pathway may lead to and/or exaggerate autoimmune responses. ICOS is induced on the cell surface after T cell activation. Similarly, ICOSL is up-regulated on APCs by several mitogenic stimuli. However, the mechanism regulating expression of the ICOS-ICOSL pair, and the significance of controlling their expression for an appropriate immune response, is largely unknown. To gain a better understanding of the importance of fine control of the ICOS-ICOSL costimulatory pathway, we generated ICOS-transgenic (Tg) mice that have high constitutive expression of ICOS in all T cells. Using ICOS-Tg mice, we studied whether in vivo immune responses were affected. Unexpectedly, we first found that ICOS-Tg mice exhibited a phenotype resembling ICOS-deficient mice in their Ag-specific Ab response, such as a defect in class switch recombination. Further examination revealed that ICOSL expression of APCs was significantly suppressed in ICOS-Tg mice. Interestingly, suppression of ICOSL was induced by interaction of ICOSL with ICOS, and it seemed to be regulated at the posttranscriptional level. The suppressive effect of the ICOS-ICOSL interaction overcame the positive effect of CD40 or B cell activation factor of the TNF family (BAFF) stimulation on ICOSL expression. Together, our studies demonstrate a novel mechanism for the regulation of ICOSL expression in vivo and suggest that the ICOS costimulatory pathway is subject to negative feedback regulation by ICOSL down-regulation in response to ICOS expression. The Journal of Immunology, 2008, 180:.
65. Katsuhiko Hayashi, Susana Lopes, Masahiro Kaneda, Fuchou Tang, Petra Hajkova, Kai Lao, Donal O'Carroll, Path Das, Alexander Tarakhovsky, Eric Miska, Azim Surani, MicroRNA Biogenesis Is Required for Mouse Primordial Germ Cell Development and Spermatogenesis, 10.1371/journal.pone.0001738, 3 , 3 , e1738 , 2008.03, Background: MicroRNAs (miRNAs) are critical regulators of transcriptional and post-transcriptional gene silencing, which are involved in multiple developmental processes in many organisms. Apart from miRNAs, mouse germ cells express another type of small RNA, piwi-interacting RNAs (piRNAs). Although it has been clear that piRNAs play a role in repression of retrotransposons during spermatogenesis, the function of miRNA in mouse germ cells has been unclear.

Methodology/Principal Findings: In this study, we first revealed the expression pattern of miRNAs by using a real-time PCR-based 220-plex miRNA expression profiling method. During development of germ cells, miR-17-92 cluster, which is thought to promote cell cycling, and the ES cell-specific cluster encoding miR-290 to -295 (miR-290-295 cluster) were highly expressed in primordial germ cells (PGCs) and spermatogonia. A set of miRNAs was developmentally regulated. We next analysed function of miRNA biogenesis in germ cell development by using conditional Dicer-knockout mice in which Dicer gene was deleted specifically in the germ cells. Dicer-deleted PGCs and spermatogonia exhibited poor proliferation. Retrotransposon activity was unexpectedly suppressed in Dicer-deleted PGCs, but not affected in the spermatogonia. In Dicer-deleted testis, spermatogenesis was retarded at an early stage when proliferation and/or early differentiation. Additionally, we analysed spermatogenesis in conditional Argonaute2-deficient mice. In contrast to Dicer-deficient testis, spermatogenesis in Argonaute2-deficient testis was indistinguishable from that in wild type.

Conclusion/Significance: These results illustrate that miRNAs are important for the proliferation of PGCs and spermatogonia, but dispensable for the repression of retrotransposons in developing germ cells. Consistently, miRNAs promoting cell cycling are highly expressed in PGCs and spermatogonia. Furthermore, based on normal spermatogenesis in Argonaute2-deficient testis, the critical function of Dicer in spermatogenesis is independent of Argonaute2..
66. Shinya Hidano, Hiroki Sasanuma, Keiko Ohshima, Ken Ichiro Seino, Lalit Kumar, Katsuhiko Hayashi, Masaki Hikida, Tomohiro Kurosaki, Masaru Taniguchi, Raif S. Geha, Daisuke Kitamura, Ryo Goitsuka, Distinct regulatory functions of SLP-76 and MIST in NK cell cytotoxicity and IFN-γ production, International Immunology, 10.1093/intimm/dxm150, 20, 3, 345-352, 2008.03, Activation of NK cells is triggered by multiple receptors. We demonstrate here that SLP-76 is required for CD16- and NKG2D-mediated NK cell cytotoxicity, while MIST negatively regulates these responses in an SLP-76-dependent manner. Exceptionally, MIST acts as a positive regulator of cytotoxicity against YAC-1 cells, although SLP-76 plays a more key role. SLP-76 acts as a dominant positive regulator for both NKG2D-mediated and YAC-1 cell-triggered IFN-γ production. Although NKG2D-mediated IFN-γ production depends on phospholipase C (PLC) γ2, YAC-1 cell-triggered IFN-γ production is PLCγ2- and Syk/ZAP-70 independent and nuclear factor-kappa B mediated. SLP-76 is required for this process in the presence of MIST but is dispensable in the absence of MIST. Thus, YAC-1 cell-triggered NKG2D-independent IFN-γ production appears to be regulated by SLP-76-dependent and -independent pathways, in which the latter is negatively regulated by MIST. Taken together, these results suggest that SLP-76 and MIST distinctly but interactively regulate NK cell cytotoxicity and IFN-γ production..
67. Susana M. Chuva De Sousa Lopes, Katsuhiko Hayashi, Tanya C. Shovlin, Will Mifsud, M. Azim Surani, Anne McLaren, X chromosome activity in mouse XX primordial germ cells, PLoS genetics, 10.1371/journal.pgen.0040030, 4, 2, 2008.02, In the early epiblast of female mice, one of the two X chromosomes is randomly inactivated by a Xist-dependent mechanism, involving the recruitment of Ezh2-Eed and the subsequent trimethylation of histone 3 on lysine 27 (H3K27me3). We demonstrate that this random inactivation process applies also to the primordial germ cell (PGC) precursors, located in the proximal region of the epiblast. PGC specification occurs at about embryonic day (E)7.5, in the extraembryonic mesoderm, after which the germ cells enter the endoderm of the invaginating hindgut. As they migrate towards the site of the future gonads, the XX PGCs gradually lose the H3K27me3 accumulation on the silent X chromosome. However, using a GFP transgene inserted into the X chromosome, we observed that the XX gonadal environment (independently of the gender) is important for the substantial reactivation of the inactive X chromosome between E11.5 and E13.5, but is not required for X-chromosome reactivation during the derivation of pluripotent embryonic germ cells. We describe in detail one of the key events during female PGC development, the epigenetic reprogramming of the X chromosome, and demonstrate the role of the XX somatic genital ridge in this process..
68. Katsuhiko Hayashi, Susana M.Chuva De Sousa Lopes, M. Azim Surani, Germ cell specification in mice, Science, 10.1126/science.1137545, 316, 5823, 394-396, 2007.04, Specification of germ cells in mice occurs relatively late in embryonic development. It is initiated by signals that induce expression of Blimp1, a key regulator of the germ cell, in a few epiblast cells of early postimplantation embryos. Blimp1 represses the incipient somatic program in these cells and promotes progression toward the germ cell fate. Blimp1 may also have a role in the maintenance of early germ cell characteristics by ensuring their escape from the somatic fate as well as possible reversion to pluripotent stem cells..
69. Y. Matsui, K. Hayashi, Epigenetic regulation for the induction of meiosis, Cellular and Molecular Life Sciences, 10.1007/s00018-006-6281-6, 64, 3, 257-262, 2007.02, The germ cell lineage is the sole cell lineage through which genomic information is transmitted into successive generations. To this end, germ cells undergo various specific differentiation steps including meiosis, whose regulation seems to correlate closely with fundamental mechanisms that create differences between germ cells and somatic cells. In mammals, meiosis is triggered by extra-embryonic stimuli such as retinoic acid, which may induce various intracellular molecular cascades promoting meiosis. In addition, the specific epigenetic status arranged in germ cells before and after induction of meiosis, including meiosis-specific transcription control based on histone methylation by a novel histone methyltransferase Meisetz, is also critical for its proper progression..
70. M. Azim Surani, Katsuhiko Hayashi, Petra Hajkova, Genetic and Epigenetic Regulators of Pluripotency, Cell, 10.1016/j.cell.2007.02.010, 128, 4, 747-762, 2007.02, Genetic and epigenetic mechanisms regulate the transition from the totipotent zygote to pluripotent primitive ectoderm cells in the inner cell mass of mouse blastocysts. These pluripotent cells can be propagated indefinitely in vitro, underpinned by a unique epigenetic state. Following implantation of the blastocyst, diverse epigenetic modifiers control differentiation of pluripotent epiblast cells into somatic cells, while specification of germ cells requires repression of the somatic program. Regenerating totipotency during development of germ cells entails re-expression of pluripotency-specific genes and extensive erasure of epigenetic modifications. Increasing knowledge of key underlying mechanisms heightens prospects for creating pluripotent cells directly from adult somatic cells..
71. Susana M.Chuva De Sousa Lopes, Katsuhiko Hayashi, M. Azim Surani, Proximal visceral endoderm and extraembryonic ectoderm regulate the formation of primordial germ cell precursors, BMC Developmental Biology, 10.1186/1471-213X-7-140, 7, 2007, Background. The extraembryonic tissues, visceral endoderm (VE) and extraembryonic ectoderm (ExE) are known to be important for the induction of primordial germ cells (PGCs) in mice via activation of the bone morphogenetic protein (BMP) signalling pathway. We investigated whether the VE and ExE have a direct role in the specification of PGCs, or in an earlier event, namely the induction of the PGC precursors in the proximal posterior epiblast cells. Results. We cultured embryonic day (E) 5.75 to E7.0 mouse embryos in an explant-assay with or without extraembryonic tissues. The reconstituted pieces of embryonic and extraembryonic tissues were assessed for the formation of both PGC precursors and specified PGCs. For this, Blimp1:gfp and Stella:gfp transgenic mouse lines were used to distinguish between PGC precursors and specified PGC, respectively. We observed that the VE regulates formation of an appropriate number of PGC precursors between E6.25-E7.25, but it is not essential for the subsequent specification of PGCs from the precursor cells. Furthermore, we show that the ExE has a different role from that of the VE, which is to restrict localization of PGC precursors to the posterior part of the embryo. Conclusion. We show that the VE and ExE have distinct roles in the induction of PGC precursors, namely the formation of a normal number of PGC precursors, and their appropriate localization during early development. However, these tissues do not have a direct role during the final stages of specification of the founder population of PGCs..
72. Mutsumi Yamamoto, Katsuhiko Hayashi, Takuya Nojima, Yumi Matsuzaki, Yohei Kawano, Hajime Karasuyama, Ryo Goitsuka, Daisuke Kitamura, BASH-novel PKC-Raf-1 pathway of pre-BCR signaling induces κ gene rearrangement, Blood, 10.1182/blood-2006-05-024968, 108, 8, 2703-2711, 2006.10, The pre-B-cell receptor (pre-BCR) is thought to signal transcriptional activation of the immunoglobulin light (L) chain gene locus, proceeding to its V-J rearrangement. The pre-BCR signaling pathway for this process is largely unknown but may involve the adaptor protein BASH (BLNK/SLP-65). Here we report that the pre-B leukemia cell lines established from affected BASH-deficient mice rearrange κL-chain gene locus and down-regulate pre-BCR upon PMA treatment or BASH reconstitution. Analyses with specific inhibitors revealed that activation of novel PKC (nPKC) and MEK, but not Ras, is necessary for the rearrangement. Accordingly, retroviral transduction of active PKCη, PKCε, or Raf-1, but not Ras, induced the κ gene rearrangement and expression in the pre-B-cell line. Tamoxifen-mediated BASH reconstitution resulted in the translocation of PKCη to the plasma membrane and κ chain expression. These data make evident that the Ras-independent BASH-nPKC-Raf-1 pathway of pre-BCR signaling induces the L-chain gene rearrangement and expression..
73. Shinya Tanaka, Jun Tsukada, Wataru Suzuki, Katsuhiko Hayashi, Kenji Tanigaki, Masayuki Tsuji, Hiromasa Inoue, Tasuku Honjo, Masato Kubo, The Interleukin-4 Enhancer CNS-2 Is Regulated by Notch Signals and Controls Initial Expression in NKT Cells and Memory-Type CD4 T Cells, Immunity, 10.1016/j.immuni.2006.04.009, 24, 6, 689-701, 2006.06, Epigenetic changes in chromatin structure at the T helper (Th2) locus correlate with interukin-4 (IL-4) and IL-13 expression during Th2 differentiation. By using a transgenic green fluorescence protein (GFP) reporter system, we show that conserved noncoding sequence-2 (CNS-2), located downstream of the Il4 locus, is a constitutively active enhancer in NKT cells as well as in a subset of CD44hi memory phenotype CD4+ T cells. CNS-2 enhancer activity and initial IL-4 expression in CD44hi CD4+ T cells were abolished in mice with a CD4-specific deletion of the transcriptional mediator of Notch signaling, Rbp-j. Depletion of CNS-2 active CD4+ T cells markedly decreased Th2 differentiation from naive CD4 T cells and antigen-specific IgE production after in vivo priming. These findings indicate that Notch-regulated CNS-2 enhancer controls initial IL-4 expression in NKT and memory phenotype CD4+ T cells and that CNS-2 active CD44hi memory phenotype T cells are important in facilitating Th2 differentiation of naive CD4+ T cells in allergic responses..
74. Takuya Nojima, Katsuhiko Hayashi, Ryo Goitsuka, Keiko Nakayama, Keiichi Nakayama, Daisuke Kitamura, Double knockout mice show BASH and PKCδ have different epistatic relationships in B cell maturation and CD40-mediated activation, Immunology Letters, 10.1016/j.imlet.2005.12.004, 105, 1, 48-54, 2006.05, The development and survival of mature B cells requires an antigen-independent signal from the B cell receptor (BCR) through an adaptor protein containing an SH2 domain, BASH (BLNK/SLP-65). It also requires signaling through BAFF and the BAFF receptor (BAFF-R), and is negatively regulated by protein kinase Cδ (PKCδ). In PKCδ-deficient mice, B cell maturation occurs independently of the BAFF receptor (BAFF-R), indicating that BAFF-R signaling promotes maturation by inhibiting the negative function of PKCδ. To clarify which of the two signaling pathways plays the primary role in B cell maturation, we crossed BASH-deficient mice with PKCδ-deficient mice to generate BASH/PKCδ-double knockout (DKO) mice. In the DKO mice, B cell maturation was blocked at the transitional type 1 (T1) stage and B cells were prone to apoptosis, in common with BASH-deficient mice. This indicates that BASH-mediated BCR signaling primarily controls B cell survival and maturation, with BAFF-R signaling and its inhibition of PKCδ acting as a secondary regulator. By contrast, CD40-mediated proliferation and antibody production, which are low in BASH-deficient mice, were rescued in the DKO mice, indicating that the suppression of CD40-mediated B cell activation by PKCδ is epistatic to BASH-mediated promotion. The physiological relevance of these opposing hierarchical effects of BASH and PKCδ in the regulation of B cell maturation and activation is discussed..
75. Hiroki Sasanuma, Akiko Tatsuno, Shinya Hidano, Keiko Ohshima, Yumi Matsuzaki, Katsuhiko Hayashi, Clifford A. Lowell, Daisuke Kitamura, Ryo Goitsuka, Dual function for the adaptor MIST in IFN-γ production by NK and CD4+NKT cells regulated by the Src kinase Fgr, Blood, 10.1182/blood-2005-10-4102, 107, 9, 3647-3655, 2006.05, Natural killer (NK) cells and NKT cells play critical early roles in host defense. Here we show that MIST, an adaptor protein belonging to the SLP-76 family, functions negatively in NK cells but positively in CD4+NKT cells. NK-cell receptor-mediated IFN-γ production was enhanced in NK cells, whereas TCR- or NK-cell receptor-mediated cytokine production was reduced in CD4+NKT cells from MIST-deficient mice. These opposite effects of MIST paralleled the exclusive expression of the Src family kinase, Fgr, in NK cells between the 2 cell populations. We further demonstrated that interaction of MIST with Fgr, mediated by the C-terminal proline-rich region of MIST and the SH3 domain of Fgr, was required for the suppression of NK-cell receptor-induced IFN-γ production. This functional interdependence of signaling molecules demonstrates a new mechanism by which adaptor proteins can act as molecular switches to control diverse responses in different cell populations..
76. Katsuhiko Hayashi, Yasuhisa Matsui, Meisetz, a novel histone tri-methyltransferase, regulates meiosis-specific epigenesis, Cell Cycle, 10.4161/cc.5.6.2572, 5, 6, 615-620, 2006.03, Meiosis is a specialized cell division that is essential to reduce the ploidy of the genome and to generate genomic diversity in all sexually reproducing organisms. Our recent investigation revealed that the meiosis-specific histone methyltransferase Meisetz (Meiosis-induced factor containing PR/SET domain and zinc-finger motif) played an essential role on proper progression of meiotic prophase in mouse germ cell lineage. The finding also suggests that the germ cell lineage has a meiosis-specific epigenetic status that is different from that of the somatic cell lineage. This article reviews the function of Meisetz and prospects the epigenetic background allowing proper progression of meiosis in mouse germ cells..
77. Katsuhiko Hayashi, Yasuhisa Matsui, Meiosis-specific histone methylation is essential for meiotic progression, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme, 51, 5, 441-445, 2006.01.
78. Katsuhiko Hayashi, Kayo Yoshida, Yasuhisa Matsui, A histone H3 methyltransferase controls epigenetic events required for meiotic prophase, 10.1038/nature04112, 438 , 7066 , 374-378 , 2005.11, Epigenetic modifications of histones regulate gene expression and chromatin structure(1,2). Here we show that Meisetz (meiosis-induced factor containing a PR/SET domain and zinc-finger motif) is a histone methyltransferase that is important for the progression of early meiotic prophase. Meisetz transcripts are detected only in germ cells entering meiotic prophase in female fetal gonads and in postnatal testis. Notably, Meisetz has catalytic activity for trimethylation, but not mono- or dimethylation, of lysine 4 of histone H3, and a transactivation activity that depends on its methylation activity. Mice in which the Meisetz gene is disrupted show sterility in both sexes due to severe impairment of the double-stranded break repair pathway, deficient pairing of homologous chromosomes and impaired sex body formation. In Meisetz-deficient testis, trimethylation of lysine 4 of histone H3 is attenuated and meiotic gene transcription is altered. These findings indicate that meiosis-specific epigenetic events in mammals are crucial for proper meiotic progression..
79. Ryoji Fujimaki, Katsuhiko Hayashi, Naoko Watanabe, Taketo Yamada, Yoshiaki Toyama, Ken Ichi Tezuka, Nobumichi Hozumi, Expression of Cre recombinase in the mouse developing chondrocytes driven by the mouse α2(XI) collagen promoter, Journal of Bone and Mineral Metabolism, 10.1007/s00774-004-0595-y, 23, 3, 270-273, 2005.05, Spatial and temporal gene inactivation by the cre-loxP system is a powerful tool for analyzing genes of interests. We generated a transgenic mouse line that expresses Cre recombinase under the control of the -742-bp promoter sequence of the mouse α2(XI) collagen gene. Crossing this mouse line with a reporter mouse revealed that Cre recombinase activity is observed specifically in developing chondrocytes. This chondrocyte-specific Cre expression was observed from the early stage of chondrocyte differentiation in forelimb at 12.5 dpc, but not expressed in mesenchymal condensations. This transgenic mouse line will be a suitable resource for the analysis of gene function in differentiating chondrocytes and the mechanism of endochondral ossification..
80. Yoshiyuki Seki, Katsuhiko Hayashi, Kunihiko Itoh, Michinao Mizugaki, Mitinori Saitou, Yasuhisa Matsui, Extensive and orderly reprogramming of genome-wide chromatin modifications associated with specification and early development of germ cells in mice, Developmental Biology, 10.1016/j.ydbio.2004.11.025, 278, 2, 440-458, 2005.02, Induction of mouse germ cells occurs from the proximal epiblast at around embryonic day (E) 7.0. These germ cells then migrate to, and enter the gonads at about E10.5 after which they undergo epigenetic reprogramming including erasure of parental imprints. However, the epigenetic properties acquired by nascent germ cells and the potential remodeling of these epigenetic marks in the subsequent migratory period have been largely unexplored. Here we have used immunohistochemistry to examine several genome-wide epigenetic modifications occurring in germ cells from their specification to their colonization of the genital ridges. We show that at around E8.0, germ cells concomitantly and significantly reduce H3-K9 dimethylation and DNA methylation, two major repressive modifications for gene expression. These events are preceded by the transient loss of all the DNA methyltransferases from their nuclei. By contrast, germ cells substantially increase the levels of H3-K27 trimethylation, another repressive modification with more plasticity, at E8.5-9.0 and maintain this state until at least E12.5. H3-K4 methylation and H3-K9 acetylation, modifications associated with transcriptionally permissive/active chromatin, are similar in germ and surrounding somatic cells but germ cells transiently increase these marks sharply upon their entry into the genital ridge. H3-K9 trimethylation, a hallmark of centromeric heterochromatin, is kept relatively constant during the periods examined. We suggest that this orderly and extensive epigenetic reprogramming in premigratory and migratory germ cells might be necessary for their reacquisition of underlying totipotency, for subsequent specific epigenetic remodeling, including the resetting of parental imprints, and for the production of gametes with an appropriate epigenotype for supporting normal development..
81. Daiji Okamura, Katsuhiko Hayashi, Yasuhisa Matsui, Mouse epiblasts change responsiveness to BMP4 signal required for PGC formation through functions of extraembryonic ectoderm, Molecular Reproduction and Development, 10.1002/mrd.20136, 70, 1, 20-29, 2005.01, Mouse primordial germ cells (PGCs) are initially identified as a cluster of alkaline phosphatase (AP)-positive cells within the extraembryonic mesoderm near the posterior part of the primitive streak at embryonic day (E) 7.25. Clonal analysis of epiblast cells has revealed that the putative precursors of PGCs are localized in the proximal epiblast, and we demonstrated that the conditions required for PGC formation are induced in the proximal region of epiblasts by extraembryonic ectoderm. Bone morphogenetic protein (BMP) 4 and BMP8b, which belong to the transforming growth factor-β (TGF-β) superfamily, might generate induction signals from extraembryonic ectoderm. Smad1 and Smad5, which are intracellular signaling molecules for BMP4, might also play a critical role in stimulating epiblasts to form PGC. However, how pluripotential epiblasts temporally and spatially respond to BMP signals to form PGCs remains unclear. The present study examines changes of responsiveness to BMP4 for PGC formation in epiblasts and their molecular mechanisms. We initially examined the effect of recombinant human (rh) BMP4 upon cultured epiblasts at different developmental stages, and found that they acquire the ability to respond to BMP4 signals for PGC formation between E5.25 and E5.5. In addition, such competence was conferred upon epiblasts by the extraembryonic ectoderm. We also showed that the increased expression of Smad1 and the onset of Smad5 expression induced by extraembryonic ectoderm might be responsible for quick acquisition of this competence. Furthermore, we show that only proximal epiblast cells maintain responsiveness to BMP4 for PGC formation at E6.0, and that this is associated with the proximal epiblast-specific expression of Smad5. These results explain why only the proximal region of epiblasts can sustain the ability to form PGCs..
82. Katsuhiko Hayashi, Takuya Nojima, Ryo Goitsuka, Daisuke Kitamura, Impaired receptor editing in the primary B cell repertoire of BASH-deficient mice, Journal of Immunology, 10.4049/jimmunol.173.10.5980, 173, 10, 5980-5988, 2004.11, The editing of B cell Ag receptor (BCR) through successive rearrangements of Ig genes has been considered to be a major mechanism for the central B cell tolerance, which precludes appearance of self-reactive B cells, through studies using anti-self-Ig transgenic/knock-in mouse systems. However, contribution of the receptor editing in the development of the normal B cell repertoire remains unclear. In addition, the signaling pathway directing this event is unknown. In this study, we demonstrate that receptor editing in anti-DNA Ig knock-in mice is impaired in the absence of an adaptor protein BASH (BLNK/SLP-65) that is involved in BCR signaling. Remarkably, the supposed hallmarks of receptor editing such as Igλ chain expression, recombination sequence rearrangements at Igκ loci, and presence of in-frame VκJκ joins in the Igκ loci inactivated by the recombination sequence rearrangements, were all diminished in BASH-deficient mice with unmanipulated Ig loci. BCR ligation-induced Igλ gene recombination in vitro was also impaired in BASH-deficient B cells. Furthermore, the BASH-deficient mice showed an excessive Ab response to a DNA carrier immunization, suggesting the presence of unedited DNA-reactive B cells in the periphery. These results not only define a signaling pathway required for receptor editing but indicate that the BCR-signaled receptor editing indeed operates in the development of normal B cell repertoire and contributes to establishing the B cell tolerance..
83. Mutsumi Yamamoto, Takuya Nojima, Katsuhiko Hayashi, Ryo Goitsuka, Koji Furukawa, Takachika Azuma, Daisuke Kitamura, BASH-deficient mice
Limited primary repertoire and antibody formation, but sufficient affinity maturation and memory B cell generation, in anti-NP response, International immunology, 10.1093/intimm/dxh116, 16, 8, 1161-1171, 2004.08, Signaling through the B cell antigen receptor (BCR) induces activation and proliferation of B cells, a response that requires the adaptor protein BASH (also known as BLNK/SLP-65). Although BASH and other molecules, such as Btk, PLCγ2 and PKCβ, are known to be essential for T cell-independent immune responses in vivo, their requirement during T cell-dependent immune responses, especially their role in antibody affinity-maturation and memory B cell generation remains unclear. In this study, we examined primary and memory immune responses to the T cell-dependent hapten antigen, (4-hydroxy-3-nitrophenyl)acetyl (NP) conjugated to chicken gammaglobulin (CGG), in BASH-deficient mice on a C57BL/6 background. In the primary response, NP-specific IgM was barely produced and the typical anti-NP IgG1/λ production was markedly attenuated, but κ chain was unexpectedly overrepresented in the anti-NP antibodies. In contrast, CGG-specific IgG1 was normally produced. In the memory response, IgG1/λ antibody with high affinity to NP was produced at normal level in the mutant mice. The frequency and distribution of somatic mutations in the VH186.2 genes of the anti-NP IgG1/λ antibody were also normal. These results indicate that BASH-mediated BCR signaling is dispensable for somatic hypermutation and affinity selection, as well as generation and response of memory B cells. Interestingly, mutated VH genes with the same clonal origin were prominent in the anti-NP antibodies of BASH-deficient mice, indicating that a limited number of original clones had been recruited into the memory compartment. Thus, the scarcity of specific clones in the primary repertoire and an impaired primary response is not detrimental to the quality and quantity of a memory response..
84. Noriyasu Seki, Mayumi Miyazaki, Wataru Suzuki, Katsuhiko Hayashi, Kazuhiko Arima, Elmarie Myburgh, Kenji Izuhara, Frank Brombacher, Masato Kubo, IL-4-Induced GATA-3 Expression Is a Time-Restricted Instruction Switch for Th2 Cell Differentiation, Journal of Immunology, 10.4049/jimmunol.172.10.6158, 172, 10, 6158-6166, 2004.05, An initial activation signal via the TCR in a restricted cytokine environment is critical for the onset of Th cell development. Cytokines regulate the expression of key transcriptional factors, T-bet and GATA-3, which instruct the direction of Th1 and Th2 differentiation, through changes in chromatin conformation. In this study, we investigated the kinetics of IL-4-mediated signaling in a transgenic mouse, expressing human IL-4R on a mouse IL-4αRR-deficient background. These experiments, allowing induction with human IL-4 at defined times, demonstrated that an IL-4 signal was required at the early stage of TCR-mediated T cell activation for lineage commitment to Th2, along with structural changes in chromatin, which take place in the conserved non-coding sequence-1 and -2 within the IL-4 locus. At later times, however, ILL-4 failed to promote efficient Th2 differentiation and decondensation of chromatin, even though GATA-3 was dearly induced in the nuclei by EL-4 stimulation. Moreover, EL-4-mediated Th2 instruction was independent from cell division mediated by initial TCR stimulation. The role of IL-4 signaling may have a time restriction during Th2 differentiation. In late stages of initial T cell activation, the chromatin structure of the IL-4 locus retains condensation state. These results demonstrate that IL-4-induced GATA-3 expression is time-restriction switch for Th2 differentiation..
85. Yoh Ichi Seki, Hiromasa Inoue, Naoko Nagata, Katsuhiko Hayashi, Satoru Fukuyama, Koichiro Matsumoto, Okiru Komine, Shinjiro Hamano, Kunisuke Himeno, Kyoko Inagaki-Ohara, Nicholas Cacalano, Anne O'Garra, Tadahilo Oshida, Hirohisa Saito, James A. Johnston, Akihiko Yoshimura, Masato Kubo, SOCS-3 regulates onset and maintenance of TH2-mediated allergic responses, Nature medicine, 10.1038/nm896, 9, 8, 1047-1054, 2003.08, Members of the suppressor of cytokine signaling (SOCS) family are involved in the pathogenesis of many inflammatory diseases. SOCS-3 is predominantly expressed in T-helper type 2 (TH2) cells, but its role in T H2-related allergic diseases remains to be investigated. In this study we provide a strong correlation between SOCS-3 expression and the pathology of asthma and atopic dermatitis, as well as serum IgE levels in allergic human patients. SOCS-3 transgenic mice showed increased TH2 responses and multiple pathological features characteristic of asthma in an airway hypersensitivity model system. In contrast, dominant-negative mutant SOCS-3 transgenic mice, as well as mice with a heterozygous deletion of Socs3, had decreased TH2 development. These data indicate that SOCS-3 has an important role in regulating the onset and maintenance of T H2-mediated allergic immune disease, and suggest that SOCS-3 may be a new therapeutic target for the development of antiallergic drugs..
86. Katsuhiko Hayashi, Mutsumi Yamamoto, Takuya Nojima, Ryo Goitsuka, Daisuke Kitamura, Distinct signaling requirements for D mu selection, IgH allelic exclusion, pre-B cell transition, and tumor suppression in B cell progenitors, 10.1016/S1074-7613(03)00142-0, 18 , 6 , 825-836 , 2003.06, The pre-B cell receptor triggers expansion and differentiation of pre-B cells (the pre-B cell transition), as well as inhibition Of V-H to DJ(H) recombination (allelic exclusion). The latter also accounts for counter-selection of pro-B cells expressing Dmu protein (Dmu selection). However, the signaling pathways responsible for these events remain poorly defined. Here we show complete arrest of B cell development at the pre-B cell transition in BASH/CD19 double mutant mice, indicating partial redundancy of the two B cell-specific adaptors. Allelic exclusion remained intact in the double mutant mice, whereas Dmu, selection was abolished in BASH mutant mice. Thus, distinct signals are required for these events. In addition, both mutant mice succumbed to pre-B cell leukemia, indicating that BASH and CD19 contribute to tumor suppression..
87. Sachiko Johmura, Masatsugu Oh-hora, Kazunori Inabe, Yumiko Nishikawa, Katsuhiko Hayashi, Elena Vigorito, Daisuke Kitamura, Martin Turner, Koh Shingu, Masaki Hikida, Tomohiro Kurosaki, Regulation of Vav localization in membrane rafts by adaptor molecules Grb2 and BLNK, Immunity, 10.1016/S1074-7613(03)00139-0, 18, 6, 777-787, 2003.06, Despite the importance of the Vav family proteins for B cell receptor (BCR) signaling, their activation mechanisms remain poorly understood. We demonstrate here that adaptor molecules Grb2 and BLNK, in addition to Vav, are required for efficient Rac1 activation in response to BCR stimulation. Loss of either Grb2 or BLNK results in decreased translocation of Vav3 to membrane rafts. By expression of Vav3 as a raft-targeted construct, the defective Rac1 activation in Grb2- or BLNK-deficient B cells is restored. Hence, our findings suggest that Grb2 and BLNK cooperate to localize Vav into membrane rafts, thereby contributing to optimal activation of Vav in B cells..
88. Akira Matsumoto, Yoh ichi Seki, Ryosuke Watanabe, Katsuhiko Hayashi, James A. Johnston, Yohsuke Harada, Ryo Abe, Akihiko Yoshimura, Masato Kubo, A role of suppressor of cytokine signaling 3 (SOCS3/CIS3/SSI3) in CD28-mediated interleukin 2 production, Journal of Experimental Medicine, 10.1084/jem.20020939, 197, 4, 425-436, 2003.02, Suppressor of cytokine signaling (SOCS)3 has been characterized as a negative feedback regulator in cytokine-mediated Janus kinase signal transducer and activator of transcription signaling. However, this study shows that T cells from transgenic mice expressing SOCS3 exhibit a significant reduction in interleukin (IL)-2 production induced by T cell receptor cross-linking when T cells are costimulated with CD28. Decreased protein expression in SOCS3+/- mice enhanced CD28-mediated IL-2 production, clearly indicating the correlation between expression level of SOCS3 and IL-2 production ability. The SOCS3 protein interacted with phosphorylated CD28 through its SH2 domain but not the kinase inhibitory region. In addition, a point mutation in the SOCS3 SH2 domain attenuated the inhibition of CD28 function in IL-2 promoter activation. Committed T helper (Th)2 cells exclusively expressed SOCS3 and production of Th2 cytokines, such as IL-4 and IL-5, was much less dependent on CD28 costimulation compared with interferon γ and IL-2 production in Th1 cells. Consistent with this notion, the expression level of SOCS3 in early T cell activation influenced the ability of IL-2 production induced by CD28 costimulation. Therefore, the SOCS3 may play an alternative role in prohibiting excessive progression of CD28-mediated IL-2 production..
89. Katsuhiko Hayashi, Takashi Kobayashi, Takashi Umino, Ryo Goitsuka, Yasuhisa Matsui, Daisuke Kitamura, SMAD1 signaling is critical for initial commitment of germ cell lineage from mouse epiblast, 10.1016/S0925-4773(02)00237-X, 118, 1-2 , 99-109 , 2002.10, Commitment of the germ cell lineage during embryogenesis depends on zygotic gene expression in mammals, but little is known about the signaling molecules required for germ cell formation. Here we show that the intracellular signaling molecule SMAD1, acting downstream of bone morphogenetic protein (BMP) receptors, is required for the commitment of germ cell lineage from epiblast in early mouse embryos. Smad1 homozygous mutant embryos (Smad1 -/-) were generated by in-frame insertion of lacZ gene into an exon of the Smad1 gene. Most of the Smad1 -/- embryos contained no primordial germ cells (PGCs) and had short allantois, while histological analysis and in situ hybridization for the mesoderm marker genes revealed that early mesoderm induction was normal in those embryos. Smad1 expression was observed in epiblast and in visceral endoderm during gastrulation, while only a few alkaline phosphatase-positive PGCs at 7.5 and 8.5 days post coitum (E7.5 and E8.5) expressed Smad1. Phosphorylated SMAD proteins were localized in the proximal region of epiblast at E6.0-6.5, where the progenitors of PGCs and of allantois reside. Single-cell reverse transcription-polymerase chain reaction analysis revealed that the expression of Smad1, -5 and -8 were sporadic and mutually independent in proximal epiblast cells. We also found that BMP4-induced differentiation of PGCs from epiblast in vitro was fully dependent on the existence of phosphorylated SMAD1. These results indicate that SMAD1 signaling possesses a critical and non-redundant function in the initial commitment of the germ cell lineage. .
90. Yoh Ichi Seki, Katsuhiko Hayashi, Akira Matsumoto, Noriyasu Seki, Jun Tsukada, John Ransom, Tetsuji Naka, Tadamitsu Kishimoto, Akihiko Yoshimura, Masato Kubo, Expression of the suppressor of cytokine signaling-5 (SOCS5) negatively regulates IL-4-dependent STAT6 activation and Th2 differentiation, Proceedings of the National Academy of Sciences of the United States of America, 10.1073/pnas.202477099, 99, 20, 13003-13008, 2002.10, The development of helper T (Th) cell subsets, which secrete distinct cytokines, plays an important role in determining the type of immune response. The IL-4-mediated Janus kinase-signal transducer and activator of transcription signaling pathway is crucial for mediating Th2 cell development. Notably, this pathway is selectively impaired in Th1 cells, although the molecular basis of this impairment remains unclear. We show here that during Th1 differentiation a reduction in the association of Janus kinase 1 with the IL-4 receptor (IL-4R) correlated with the appearance of the suppressor of cytokine signaling-5 (SOCS5). SOCS5 protein was preferentially expressed in committed Th1 cells and interacted with the cytoplasmic region of the IL-4Rα chain irrespective of receptor tyrosine phosphorylation. This unconventional interaction of SOCS5 protein with the IL-4R resulted in the inhibition of IL-4-mediated signal transducer and activator of transcription-6 activation. T cells from transgenic mice constitutively expressing SOCS5 exhibited a significant reduction of IL-4-mediated Th2 development. Therefore, the induced SOCS5 protein in Th1 differentiation environment may play an important role by regulating Th1 and Th2 balance..
91. A. Terauchi, K. Hayashi, D. Kitamura, Y. Kozono, N. Motoyama, T. Azuma, A pivotal role for DNase I-sensitive regions 3b and/or 4 in the induction of somatic hypermutation of IgH genes, Journal of Immunology, 10.4049/jimmunol.167.2.811, 167, 2, 811-820, 2001.07, Chimeric mice were prepared from embryonic stem cells transfected with IgH genes as transgenes and RAG-2-deficient blastocysts for the purpose of identifying the cis-acting elements responsible for the induction of somatic hypermutation. Among the three transgene constructs used, the VH promoter, the rearranged VH-D-JH, an intron enhancer/matrix attachment region, and human Cμ were common to all, but the 3′-untranslated region in each construct was different. After immunization of mice with a T cell-dependent Ag, the distribution and frequency of hypermutation in transgenes were analyzed. The transgene lacking the 3′ untranslated region showed a marginal degree of hypermutation. Addition of the 3′ enhancer resulted in a slight increase in the number of mutations. However, the transgene containing DNase I-sensitive regions 3b and 4 in addition to the 3′ enhancer showed more than a 10-fold increase in hypermutation, reaching levels comparable to those observed in endogenous VH186.2 genes of C57BL/6 mice..
92. Y. Harada, M. Tokushima, Y. Matsumoto, S. Ogawa, M. Otsuka, K. Hayashi, B. D. Weiss, C. H. June, R. Abe, Critical requirement for the membrane-proximal cytosolic tyrosine residue for CD28-mediated costimulation in vivo, Journal of Immunology, 10.4049/jimmunol.166.6.3797, 166, 6, 3797-3803, 2001.03, The YMNM motif that exists in the CD28 cytoplasmic domain is known as a binding site for phosphatidylinositol 3-kinase and Grb-2 and is considered to be important for CD28-mediated costimulation. To address the role of the YMNM motif in CD28 cosignaling in primary T cells, we generated transgenic mice on a CD28 null background that express a CD28 mutant lacking binding ability to phosphatidylinositol 3-kinase and Grb-2. After anti-CD3 and anti-CD28 Ab stimulation in vitro, the initial proliferative response and IL-2 secretion in CD28 Y189F transgenic T cells were severely compromised, while later responses were intact. In contrast to anti-CD3 and anti-CD28 Ab stimulation, PMA and anti-CD28 Ab stimulation failed to induce IL-2 production from CD28 Y189F transgenic T cells at any time point. Using the graft-vs-host reaction system, we assessed the role of the YMNM motif for CD28-mediated costimulation in vivo and found that CD28 Y189F transgenic spleen cells failed to engraft and could not induce acute graft-vs-host reaction. Together, these results suggest that the membrane-proximal tyrosine of CD28 is required for costimulation in vivo. Furthermore, these results indicate that the results from in vitro assays of CD28-mediated costimulation may not always correlate with T cell activation in vivo..
93. Katsuhiko Hayashi, Ryo Nittono, Noriaki Okamoto, Sachiyo Tsuji, Yasushi Hara, Daisuke Kitamura, The B cell-restricted adaptor BASH is required for normal development and antigen receptor-mediated activation of B cells, 10.1073/pnas.040575697, 97, 6, 2755-2760, 2000.03, B cell antigen receptor signals development, activation, proliferation, or apoptosis of B cells depending on their condition, and its proper signaling is critical for activation and homeostasis of the immune system. The B cell-restricted adaptor protein BASH (also termed BLNK/SLP-65) is rapidly phosphorylated by the tyrosine kinase Syk after BCR ligation and binds to various signaling proteins. BASH structurally resembles SLP-76, which is essential for T cell development and T cell receptor signaling. To evaluate the role for BASH in B cell development and function in vivo, we disrupted BASH alleles in embryonic stem cells by means of homologous recombination and used these cells to complement lymphocyte-incompetent blastocysts from RAG2-deficient mice. In the resultant chimeric mice, T cell development was apparently normal, but B cell development was impaired, and a normally rare population of large preB cells expressing preB cell receptor dominated in the bone marrow in place of small preB cells, although they were mostly noncycling. In addition, the mature B cell populations in the periphery and the bone marrow profoundly decreased in size, as did B-l cells in the peritoneal cavity, and serum lg was severely reduced. The BASH-deficient B cells scarcely proliferated or up-regulated B7-2 in response to BCR ligation and poorly proliferated upon CD40 ligation or lipopolysaccharide stimulation. This phenotype indicates that BASH is critical for preB cell receptor signaling inducing proliferation of large preB cells and the following differentiation. for peripheral B cell maturation, and for BCR signaling inducing activation/proliferation of B cells..
94. Ryo Goitsuka, Hideki Kanazashi, Hiroki Sasanuma, Yu ichi Fujimura, Yuri Hidaka, Akiko Tatsuno, Chisei Ra, Katsuhiko Hayashi, Daisuke Kitamura, A BASH/SLP-76-related adaptor protein MIST/Clnk involved in IgE receptor-mediated mast cell degranulation, International Immunology, 10.1093/intimm/12.4.573, 12, 4, 573-580, 2000.01, Cross-linking of the high-affinity IgE receptor (FcεRI) on mast cells by IgE-antigen complex triggers signal transduction cascades leading to the release of inflammatory mediators and production of cytokines, which are critical for the development of allergic reactions. We have identified a novel member of the BASH/SLP-76 immunoreceptor-coupled adaptor family expressed in mast cells, termed MIST (for mast cell immunoreceptor signal transducer), which has later been found to be identical to a recently reported cytokine-dependent hemopoietic cell linker, Clnk. Upon FcεRI cross-linking, MIST/Clnk is tyrosine phosphorylated and associates with signaling proteins, phospholipase Cγ, Vav, Grb2 and linker for activation of T cells (LAT). Overexpression of a mutant form of MIST/Clnk inhibited FcεRI-mediated degranulation, increase in intracellular Ca2+, NF-AT activation and phosphorylation of LAT. As a crucial signaling component for FcεRI-induced mast cell degranulation, MIST/Clnk might serve as a target for anti-allergic therapy..