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Dive into the research topics where Hiroki Danno is active.

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Featured researches published by Hiroki Danno.


Genome Biology | 2013

Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expression heterogeneity

Yohei Sasagawa; Itoshi Nikaido; Tetsutaro Hayashi; Hiroki Danno; Kenichiro D. Uno; Takeshi Imai; Hiroki R. Ueda

Development of a highly reproducible and sensitive single-cell RNA sequencing (RNA-seq) method would facilitate the understanding of the biological roles and underlying mechanisms of non-genetic cellular heterogeneity. In this study, we report a novel single-cell RNA-seq method called Quartz-Seq that has a simpler protocol and higher reproducibility and sensitivity than existing methods. We show that single-cell Quartz-Seq can quantitatively detect various kinds of non-genetic cellular heterogeneity, and can detect different cell types and different cell-cycle phases of a single cell type. Moreover, this method can comprehensively reveal gene-expression heterogeneity between single cells of the same cell type in the same cell-cycle phase.


Proceedings of the National Academy of Sciences of the United States of America | 2008

Molecular links among the causative genes for ocular malformation: Otx2 and Sox2 coregulate Rax expression

Hiroki Danno; Tatsuo Michiue; Keisuke Hitachi; Akira Yukita; Shoichi Ishiura; Makoto Asashima

The neural-related genes Sox2, Pax6, Otx2, and Rax have been associated with severe ocular malformations such as anophthalmia and microphthalmia, but it remains unclear as to how these genes are linked functionally. We analyzed the upstream signaling of Xenopus Rax (also known as Rx1) and identified the Otx2 and Sox2 proteins as direct upstream regulators of Rax. We revealed that endogenous Otx2 and Sox2 proteins bound to the conserved noncoding sequence (CNS1) located ≈2 kb upstream of the Rax promoter. This sequence is conserved among vertebrates and is required for potent transcriptional activity. Reporter assays showed that Otx2 and Sox2 synergistically activated transcription via CNS1. Furthermore, the Otx2 and Sox2 proteins physically interacted with each other, and this interaction was affected by the Sox2-missense mutations identified in these ocular disorders. These results demonstrate that the direct interaction and interdependence between the Otx2 and Sox2 proteins coordinate Rax expression in eye development, providing molecular linkages among the genes responsible for ocular malformation.


In Vitro Cellular & Developmental Biology – Animal | 2010

BMP4 induction of trophoblast from mouse embryonic stem cells in defined culture conditions on laminin.

Yohei Hayashi; Miho K. Furue; Satoshi Tanaka; Michiko Hirose; Noriko Wakisaka; Hiroki Danno; Kiyoshi Ohnuma; Shiho Oeda; Yuko Aihara; Kunio Shiota; Atsuo Ogura; Shoichi Ishiura; Makoto Asashima

Because mouse embryonic stem cells (mESCs) do not contribute to the formation of extraembryonic placenta when they are injected into blastocysts, it is believed that mESCs do not differentiate into trophoblast whereas human embryonic stem cells (hESCs) can express trophoblast markers when exposed to bone morphogenetic protein 4 (BMP4) in vitro. To test whether mESCs have the potential to differentiate into trophoblast, we assessed the effect of BMP4 on mESCs in a defined monolayer culture condition. The expression of trophoblast-specific transcription factors such as Cdx2, Dlx3, Esx1, Gata3, Hand1, Mash2, and Plx1 was specifically upregulated in the BMP4-treated differentiated cells, and these cells expressed trophoblast markers. These results suggest that BMP4 treatment in defined culture conditions enabled mESCs to differentiate into trophoblast. This differentiation was inhibited by serum or leukemia inhibitory factor, which are generally used for mESC culture. In addition, we studied the mechanism underlying BMP4-directed mESC differentiation into trophoblast. Our results showed that BMP4 activates the Smad pathway in mESCs inducing Cdx2 expression, which plays a crucial role in trophoblast differentiation, through the binding of Smad protein to the Cdx2 genomic enhancer sequence. Our findings imply that there is a common molecular mechanism underlying hESC and mESC differentiation into trophoblast.


Developmental Dynamics | 2004

XIdax, an inhibitor of the canonical Wnt pathway, is required for anterior neural structure formation in Xenopus

Tatsuo Michiue; Akimasa Fukui; Akira Yukita; Kenji Sakurai; Hiroki Danno; Akira Kikuchi; Makoto Asashima

Wnt signaling pathways are involved during various stages in the development of many species. In Xenopus, the accumulation of β‐catenin on the dorsal side of embryo is required for induction of the organizer, while the head structure formation requires inhibition of Wnt signaling. Here, we report a role for xIdax, a negative regulator of Wnt signaling. XIdax is expressed in neural tissues at the neurula stage, and in the restricted region of the tadpole brain. Ectopic expression of xIdax inhibits the target gene expression, suggesting that xIdax can inhibit canonical Wnt signaling. To examine the function of xIdax, a morpholino oligo for xIdax (xIdaxMO) was designed. An injection into an animal pole cell caused a loss of forebrain. The anterior neural marker expression is decreased in xIdaxMO‐injected embryo, suggesting that xIdax is required for anterior neural development. Moreover, a negative regulator that acts downstream of xIdax rescued this defect. We propose that Idax functions are dependent on the canonical Wnt pathway and are crucial for the anterior neural development. Developmental Dynamics 230:79–90, 2004.


The International Journal of Developmental Biology | 2009

The Xenopus Bowline/Ripply family proteins negatively regulate the transcriptional activity of T-box transcription factors

Keisuke Hitachi; Hiroki Danno; Shunsuke Tazumi; Yuko Aihara; Hideho Uchiyama; Koji Okabayashi; Akiko Kondow; Makoto Asashima

Bowline, which is a member of the Xenopus Bowline/Ripply family of proteins, represses the transcription of somitogenesis-related genes before somite segmentation, which makes Bowline indispensable for somitogenesis. Although there are three bowline/Ripply family genes in each vertebrate species, it is not known whether the Bowline/Ripply family proteins share a common role in development. To elucidate their developmental roles, we examined the expression patterns and functions of the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline, and a novel member of this protein family, xRipply3. We found that the expression patterns of bowline and ledgerline overlapped in the presomitic mesoderm (PSM), whereas ledgerline was additionally expressed in the newly formed somites. In addition, we isolated xRipply3, which is expressed in the pharyngeal region. Co-immunoprecipitation assays revealed that Ledgerline and xRipply3 interacted with T-box proteins and the transcriptional co-repressor Groucho/TLE. In luciferase assays, xRipply3 weakly suppressed the transcriptional activity of Tbx1, while Ledgerline strongly suppressed that of Tbx6. In line with the repressive role of Ledgerline, knockdown of Ledgerline resulted in enlargement of expression regions of the somitogenesis-related-genes mespb and Tbx6. Inhibition of histone deacetylase activity increased the expression of mespb, as seen in the Bowline and Ledgerline knockdown experiments. These results suggest that the Groucho-HDAC complex is required for the repressive activity of Bowline/Ripply family proteins during Xenopus somitogenesis. We conclude that although the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline and xRipply3 are expressed differentially, they all act as negative regulators of T-box proteins.


Genes to Cells | 2008

Nucleoredoxin regulates the Wnt/planar cell polarity pathway in Xenopus

Yosuke Funato; Tatsuo Michiue; Takeshi Terabayashi; Akira Yukita; Hiroki Danno; Makoto Asashima; Hiroaki Miki

The Wnt signaling pathway is conserved across species, and is essential for early development. We previously identified nucleoredoxin (NRX) as a protein that interacts with dishevelled (Dvl) in vivo to negatively regulate the Wnt/β‐catenin pathway. However, whether NRX affects another branch of the Wnt pathway, the Wnt/planar cell polarity (PCP) pathway, remains unclear. Here we show that NRX regulates the Wnt/PCP pathway. In Xenopus laevis, over‐expression or depletion of NRX by injection of NRX mRNA or antisense morpholino oligonucleotide, respectively, yields the bent‐axis phenotype that is typically observed in embryos with abnormal PCP pathway activity. In co‐injection experiments of Dvl and NRX mRNA, NRX suppresses the Dvl‐induced bent‐axis phenotype. Over‐expression or depletion of NRX also suppresses the convergent extension movements that are believed to underlie normal gastrulation. We also found that NRX can inhibit Dvl‐induced up‐regulation of c‐Jun phosphorylation. These results indicate that NRX plays crucial roles in the Wnt/PCP pathway through Dvl and regulates Xenopus gastrulation movements.


The International Journal of Developmental Biology | 2008

Retinoic acid metabolizing factor xCyp26c is specifically expressed in neuroectoderm and regulates anterior neural patterning in Xenopus laevis.

Misaki Tanibe; Tatsuo Michiue; Akira Yukita; Hiroki Danno; Masayuki Ikuzawa; Shoichi Ishiura; Makoto Asashima

Anterior-posterior neural patterning is determined during gastrulation when head structure is induced. Induction of anterior neural structures requires inhibition of Wnt signaling by several Wnt antagonists. We performed microarray analysis to isolate genes regulated by canonical Wnt signaling and abundantly expressed in the anterior neuroectoderm at the early neurula stage. We identified xCyp26c, a Cyp26 (RA-metabolizing protein)-family gene. In situ hybridization showed xCyp26c expression restricted to the anterior region of neurula, while xCyp26a was expressed in both anterior and posterior regions. At the tadpole stage, xCyp26c was also expressed in restricted sets of cranial nerves. Microarray, RT-PCR and in situ hybridization analyses revealed decreased xCyp26c expression with overexpression of beta-catenin, suggesting regulation by Wnt/beta-catenin signaling. We also assessed the effects of retinoic acid (RA) on xCyp26c expression. Embryos treated with 10(-7) M RA showed an anterior shift in the spatial expression of xCyp26c, reflecting a posteriorization effect. Conversely, expression patterns in embryos treated with more than 10(-6) M RA were less affected and remained restricted to the most anterior region. Moreover, injection of xCyp26c mRNA into animal poles caused head defects, and exogenous expression of xCyp26c rescued the posteriorizing effect of RA treatment. Taken together, these results implicated a role for xCyp26c in anterior patterning via RA signaling.


Mechanisms of Development | 2005

Novel Daple-like protein positively regulates both the Wnt/β-catenin pathway and the Wnt/JNK pathway in Xenopus

Hiroki Kobayashi; Tatsuo Michiue; Akira Yukita; Hiroki Danno; Kenji Sakurai; Akimasa Fukui; Akira Kikuchi; Makoto Asashima

Wnt signaling pathways are essential in various developmental processes including differentiation, proliferation, cell migration, and cell polarity. Wnt proteins execute their multiple functions by activating distinct intracellular signaling cascades, although the mechanisms underlying this activation are not fully understood. We identified a novel Daple-like protein in Xenopus and named it xDal (Xenopus Daple-like). As with Daple, xDal contains several leucine zipper-like regions (LZLs) and a putative PDZ domain-binding motif, and can interact directly with the dishevelled protein. In contrast to mDaple, injection of xDal mRNA into the dorso-vegetal blastomere does not induce ventralization and acted synergistically with xdsh in secondary axis induction. XDal also induced expression of siamois and xnr-3, suggesting that XDal functions as a positive regulator of the Wnt/beta-catenin pathway. Injection of xDal mRNA into the dorso-animal blastomere, however, induced gastrulation-defective phenotypes in a dose-dependent manner. In addition, xDal inhibited activin-induced elongation of animal caps and enhanced c-jun phosphorylation. Based on these findings, xDal is also thought to function in the Wnt/JNK pathway. Moreover, functional domain analysis with several deletion mutants indicated that xDal requires both a putative PDZ domain-binding motif and at least one LZL for its activity. These findings with xDal will provide new information on the Wnt signaling pathways.


Developmental Dynamics | 2007

XSUMO-1 is required for normal mesoderm induction and axis elongation during early Xenopus development

Akira Yukita; Tatsuo Michiue; Hiroki Danno; Makoto Asashima

The small ubiquitin‐related modifier (SUMO) is a member of the ubiquitin‐like protein family, and SUMO conjugation (SUMOylation) resembles ubiquitination. Despite many SUMOylation target proteins being reported, the role of this system in vertebrate development remains unclear. We inhibited the function of Xenopus SUMO‐1 (XSUMO‐1) using a morpholino antisense oligo against XSUMO‐1 (XSUMO‐1‐MO) to clarify the role of SUMOylation. XSUMO‐1‐MO inhibited normal axis formation in embryos and elongation of activin‐treated animal caps. The expression of several mesoderm markers was reduced by XSUMO‐1‐MO. We measured activin‐like activity by using a reporter construct containing a multimer of activin‐responsive elements from the Goosecoid promoter, [DE(6x)Luc]. This assay showed that XSUMO‐1‐MO directly inhibited activin/nodal signaling. Furthermore, XSUMO‐1‐MO inhibited ectopic axis formation induced by XSmad2, and XSmad2/4 mRNA could not rescue the axis elongation defect induced by XSUMO‐1‐MO. These results suggested that XSUMO‐1 is required for normal axis elongation, at least partly mediating activin/nodal signaling. Developmental Dynamics 236:2757–2766, 2007.


Journal of Experimental Zoology | 2008

Developmental Potential for Morphogenesis In Vivo and In Vitro

Kunihiko Kaneko; Katsuhiko Sato; Tatsuo Michiue; Koji Okabayashi; Kiyoshi Ohnuma; Hiroki Danno; Makoto Asashima

Development is a complex process that involves differentiation into a variety of cell types. In spite of its complexity, the macroscopic pattern and cell types are robust to environmental and developmental perturbations. Even in vitro far from normal developmental conditions, ten normal tissues have been generated from Xenopus animal caps by successive treatment with activin and retinoic acid (RA). To describe both normal development and in vitro organogenesis, we introduce developmental potential following the pioneering study by Waddington. This potential value represents changeability of a cellular state, which decreases toward a local minimum through development. The attraction to a particular cell type through development is described as a process to decrease the potential value to its local minimum. By choosing an explicit potential form as a function of the concentrations of treated activin and RA, the concentration dependence of in vitro organogenesis is reproduced. The potential landscape is shown to have several local minima, each of which represents a stable cell type. This potential also explains why the induction of given tissues requires more treatment of activin at later stages. The consequences of the developmental potential hypothesis encompass the robustness of each tissue generation, the loss of competence through development, and the order of tissues in induction by tissues, which we have confirmed experimentally for in vitro organogenesis. The developmental potential hypothesis for a global description of early development is crucial to understanding the robustness of morphogenesis and explains the achievement of in vitro organogenesis using few molecules as well.

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Makoto Asashima

National Institute of Advanced Industrial Science and Technology

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Akira Yukita

Matsumoto Dental University

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Itoshi Nikaido

Yokohama City University

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Kiyoshi Ohnuma

Nagaoka University of Technology

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