Publications by authors named "Hiroshi Kiyonari"

213 Publications

Dysfunction of the proteoglycan Tsukushi causes hydrocephalus through altered neurogenesis in the subventricular zone in mice.

Sci Transl Med 2021 Mar;13(587)

Department of Developmental Neurobiology, Graduate School of Life Sciences, Kumamoto University, 1-1-1 Honjo, Chuo-ku, Kumamoto 860-8556, Japan.

The lateral ventricle (LV) is flanked by the subventricular zone (SVZ), a neural stem cell (NSC) niche rich in extrinsic growth factors regulating NSC maintenance, proliferation, and neuronal differentiation. Dysregulation of the SVZ niche causes LV expansion, a condition known as hydrocephalus; however, the underlying pathological mechanisms are unclear. We show that deficiency of the proteoglycan Tsukushi (TSK) in ependymal cells at the LV surface and in the cerebrospinal fluid results in hydrocephalus with neurodevelopmental disorder-like symptoms in mice. These symptoms are accompanied by altered differentiation and survival of the NSC lineage, disrupted ependymal structure, and dysregulated Wnt signaling. Multiple TSK variants found in patients with hydrocephalus exhibit reduced physiological activity in mice in vivo and in vitro. Administration of wild-type TSK protein or Wnt antagonists, but not of hydrocephalus-related TSK variants, in the LV of TSK knockout mice prevented hydrocephalus and preserved SVZ neurogenesis. These observations suggest that TSK plays a crucial role as a niche molecule modulating the fate of SVZ NSCs and point to TSK as a candidate for the diagnosis and therapy of hydrocephalus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/scitranslmed.aay7896DOI Listing
March 2021

Cyst formation in proximal renal tubules caused by dysfunction of the microtubule minus-end regulator CAMSAP3.

Sci Rep 2021 Mar 12;11(1):5857. Epub 2021 Mar 12.

Laboratory of Cytoskeletal Logistics, Department of Life Science and Medical Bioscience, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsucho, Shinjuku-ku, Tokyo, 162-8480, Japan.

Epithelial cells organize an ordered array of non-centrosomal microtubules, the minus ends of which are regulated by CAMSAP3. The role of these microtubules in epithelial functions, however, is poorly understood. Here, we show that the kidneys of mice in which Camsap3 is mutated develop cysts at the proximal convoluted tubules (PCTs). PCTs were severely dilated in the mutant kidneys, and they also exhibited enhanced cell proliferation. In these PCTs, epithelial cells became flattened along with perturbation of microtubule arrays as well as of certain subcellular structures such as interdigitating basal processes. Furthermore, YAP and PIEZO1, which are known as mechanosensitive regulators for cell shaping and proliferation, were activated in these mutant PCT cells. These observations suggest that CAMSAP3-mediated microtubule networks are important for maintaining the proper mechanical properties of PCT cells, and its loss triggers cell deformation and proliferation via activation of mechanosensors, resulting in the dilation of PCTs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-021-85416-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7954811PMC
March 2021

Stepwise cell fate decision pathways during osteoclastogenesis at single-cell resolution.

Nat Metab 2020 12 7;2(12):1382-1390. Epub 2020 Dec 7.

Department of Immunology, Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan.

Osteoclasts are the exclusive bone-resorbing cells, playing a central role in bone metabolism, as well as the bone damage that occurs under pathological conditions. In postnatal life, haematopoietic stem-cell-derived precursors give rise to osteoclasts in response to stimulation with macrophage colony-stimulating factor and receptor activator of nuclear factor-κB ligand, both of which are produced by osteoclastogenesis-supporting cells such as osteoblasts and osteocytes. However, the precise mechanisms underlying cell fate specification during osteoclast differentiation remain unclear. Here, we report the transcriptional profiling of 7,228 murine cells undergoing in vitro osteoclastogenesis, describing the stepwise events that take place during the osteoclast fate decision process. Based on our single-cell transcriptomic dataset, we find that osteoclast precursor cells transiently express CD11c, and deletion of receptor activator of nuclear factor-κB specifically in CD11c-expressing cells inhibited osteoclast formation in vivo and in vitro. Furthermore, we identify Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (Cited2) as the molecular switch triggering terminal differentiation of osteoclasts, and deletion of Cited2 in osteoclast precursors in vivo resulted in a failure to commit to osteoclast fate. Together, the results of this study provide a detailed molecular road map of the osteoclast differentiation process, refining and expanding our understanding of the molecular mechanisms underlying osteoclastogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s42255-020-00318-yDOI Listing
December 2020

CAMSAP1 breaks the homeostatic microtubule network to instruct neuronal polarity.

Proc Natl Acad Sci U S A 2020 09 24;117(36):22193-22203. Epub 2020 Aug 24.

State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Innovation Academy for Seed Design, Chinese Academy of Sciences, 10019 Beijing, China;

The establishment of axon/dendrite polarity is fundamental for neurons to integrate into functional circuits, and this process is critically dependent on microtubules (MTs). In the early stages of the establishment process, MTs in axons change dramatically with the morphological building of neurons; however, how the MT network changes are triggered is unclear. Here we show that CAMSAP1 plays a decisive role in the neuronal axon identification process by regulating the number of MTs. Neurons lacking CAMSAP1 form a multiple axon phenotype in vitro, while the multipolar-bipolar transition and radial migration are blocked in vivo. We demonstrate that the polarity regulator MARK2 kinase phosphorylates CAMSAP1 and affects its ability to bind to MTs, which in turn changes the protection of MT minus-ends and also triggers asymmetric distribution of MTs. Our results indicate that the polarized MT network in neurons is a decisive factor in establishing axon/dendritic polarity and is initially triggered by polarized signals.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1913177117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486724PMC
September 2020

Pronuclear Microinjection during S-Phase Increases the Efficiency of CRISPR-Cas9-Assisted Knockin of Large DNA Donors in Mouse Zygotes.

Cell Rep 2020 05;31(7):107653

Laboratory for Animal Resources and Genetic Engineering, RIKEN Center for Biosystems Dynamics Research, 2-2-3 Minatojima Minami-machi, Chuou-ku, Kobe 650-0047, Japan. Electronic address:

In CRISPR-Cas9-assisted knockin (KI) in zygotes, a remaining challenge is routinely achieving high-efficiency KI of large (kilobase-sized) DNA elements. Here, we focus on the timing of pronuclear injection and establish a reliable homologous recombination (HR)-based method to generate large KIs in zygotes compared with two other types of KI strategies involving distinct DNA repair pathways. At the ROSA26 locus, pronuclear injection with CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and Cas9 protein at the S phase by using the HR-based method yields the most efficient and accurate KIs (up to 70%). This approach is also generally effective for generating large KI alleles at other gene loci. We further apply our method to efficiently obtain biallelic ROSA26 KIs by sequential injection into both pronuclei. Our results suggest that delivery of genome editing components and donor DNA into S-phase zygotes is critical for efficient KI of large DNA elements.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.107653DOI Listing
May 2020

Transient and lineage-restricted requirement of Ebf3 for sternum ossification.

Development 2020 05 12;147(9). Epub 2020 May 12.

Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan

Osteoblasts arise from bone-surrounding connective tissue containing tenocytes and fibroblasts. Lineages of these cell populations and mechanisms of their differentiation are not well understood. Screening enhancer-trap lines of zebrafish allowed us to identify Ebf3 as a transcription factor marking tenocytes and connective tissue cells in skeletal muscle of embryos. Knockout of Ebf3 in mice had no effect on chondrogenesis but led to sternum ossification defects as a result of defective generation of Runx2 pre-osteoblasts. Conditional and temporal Ebf3 knockout mice revealed requirements of Ebf3 in the lateral plate mesenchyme cells (LPMs), especially in tendon/muscle connective tissue cells, and a stage-specific Ebf3 requirement at embryonic day 9.5-10.5. Upregulated expression of connective tissue markers, such as Egr1/2 and Osr1, increased number of Islet1 mesenchyme cells, and downregulation of gene expression of the Runx2 regulator Shox2 in Ebf3-deleted thoracic LPMs suggest crucial roles of Ebf3 in the onset of lateral plate mesoderm differentiation towards osteoblasts forming sternum tissues.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dev.186239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7240299PMC
May 2020

Changes in Wnt-Dependent Neuronal Morphology Underlie the Anatomical Diversification of Neocortical Homologs in Amniotes.

Cell Rep 2020 05;31(5):107592

Developmental Neurobiology, Kyoto Prefectural University of Medicine, INAMORI Memorial Building, 1-5 Shimogamo-Hangi cho, Sakyoku, Kyoto 606-0823, Japan.

The six-layered neocortex is a shared characteristic of all mammals, but not of non-mammalian species, and its formation requires an inside-out pattern of neuronal migration. The extant reptilian dorsal cortex is thought to represent an ancestral form of the neocortex, although how the reptilian three-layered cortex is formed is poorly understood. Here, we show unique patterns of lamination and neuronal migration in the developing reptilian cortex. While the multipolar-to-bipolar transition of migrating neurons is essential for mammalian cortical development, the reptilian cortex lacks bipolar-shaped migrating neurons, resulting in an outside-in pattern of cortical development. Furthermore, dynamic regulation of Wnt signal strengths contributes to neuronal morphological changes, which is conserved across species. Our data preclude the idea that the six-layered mammalian neocortex emerged by simple addition to the reptilian dorsal cortex but suggest that the acquisition of a novel neuronal morphology based on conserved developmental programs contributed to neocortical evolution.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2020.107592DOI Listing
May 2020

LUBAC accelerates B-cell lymphomagenesis by conferring resistance to genotoxic stress on B cells.

Blood 2020 08;136(6):684-697

Department of Hematology and Oncology.

The linear ubiquitin chain assembly complex (LUBAC) is a key regulator of NF-κB signaling. Activating single-nucleotide polymorphisms of HOIP, the catalytic subunit of LUBAC, are enriched in patients with activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL), and expression of HOIP, which parallels LUBAC activity, is elevated in ABC-DLBCL samples. Thus, to clarify the precise roles of LUBAC in lymphomagenesis, we generated a mouse model with augmented expression of HOIP in B cells. Interestingly, augmented HOIP expression facilitated DLBCL-like B-cell lymphomagenesis driven by MYD88-activating mutation. The developed lymphoma cells partly shared somatic gene mutations with human DLBCLs, with increased frequency of a typical AID mutation pattern. In vitro analysis revealed that HOIP overexpression protected B cells from DNA damage-induced cell death through NF-κB activation, and analysis of the human DLBCL database showed that expression of HOIP positively correlated with gene signatures representing regulation of apoptosis signaling, as well as NF-κB signaling. These results indicate that HOIP facilitates lymphomagenesis by preventing cell death and augmenting NF-κB signaling, leading to accumulation of AID-mediated mutations. Furthermore, a natural compound that specifically inhibits LUBAC was shown to suppress the tumor growth in a mouse transplantation model. Collectively, our data indicate that LUBAC is crucially involved in B-cell lymphomagenesis through protection against DNA damage-induced cell death and is a suitable therapeutic target for B-cell lymphomas.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2019002654DOI Listing
August 2020

The large GTPase Rab44 regulates granule exocytosis in mast cells and IgE-mediated anaphylaxis.

Cell Mol Immunol 2020 12 1;17(12):1287-1289. Epub 2020 Apr 1.

Department of Dental Pharmacology, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, 8528588, Japan.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41423-020-0413-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7784977PMC
December 2020

Enhanced homologous recombination by the modulation of targeting vector ends.

Sci Rep 2020 02 13;10(1):2518. Epub 2020 Feb 13.

RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuou-ku, Kobe, 650-0047, Japan.

The field of genome editing was founded on the establishment of methods, such as the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR/Cas) system, used to target DNA double-strand breaks (DSBs). However, the efficiency of genome editing also largely depends on the endogenous cellular repair machinery. Here, we report that the specific modulation of targeting vectors to provide 3' overhangs at both ends increased the efficiency of homology-directed repair (HDR) in embryonic stem cells. We applied the modulated targeting vectors to produce homologous recombinant mice directly by pronuclear injection, but the frequency of HDR was low. Furthermore, we combined our method with the CRISPR/Cas9 system, resulting in a significant increase in HDR frequency. Thus, our HDR-based method, enhanced homologous recombination for genome targeting (eHOT), is a new and powerful method for genome engineering.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-58893-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7018964PMC
February 2020

Dmrt genes participate in the development of Cajal-Retzius cells derived from the cortical hem in the telencephalon.

Dev Dyn 2020 06 12;249(6):698-710. Epub 2020 Feb 12.

Department of Developmental Neuroscience, United Centers for Advanced Research and Translational Medicine (ART), Tohoku University Graduate School of Medicine, Sendai, Miyagi, Japan.

Background: During development, Cajal-Retzius (CR) cells are the first generated and essential pioneering neurons that control neuronal migration and arealization in the mammalian cortex. CR cells are derived from specific regions within the telencephalon, that is, the pallial septum in the rostromedial cortex, the pallial-subpallial boundary, and the cortical hem (CH) in the caudomedial cortex. However, the molecular mechanism underlying the generation of CR cell subtypes in distinct regions of origin is poorly understood.

Results: We found that double-sex and mab-3 related transcription factor (Dmrt) genes, that is, Dmrta1 and Dmrt3, were expressed in the progenitor domains that produce CR cells. The number of CH-derived CR cells was severely decreased in Dmrt3 mutants, especially in Dmrta1 and Dmrt3 double mutants. The reduced production of the CR cells was consistent with the developmental impairment of the CH structures in the medial telencephalon from which the CR cells are produced.

Conclusion: Dmrta1 and Dmrt3 cooperatively regulate patterning of the CH structure and production of the CR cells from the CH during cortical development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/dvdy.156DOI Listing
June 2020

Nodal paralogues underlie distinct mechanisms for visceral left-right asymmetry in reptiles and mammals.

Nat Ecol Evol 2020 02 6;4(2):261-269. Epub 2020 Jan 6.

Laboratory for Organismal Patterning, RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.

Unidirectional fluid flow generated by motile cilia at the left-right organizer (LRO) breaks left-right (L-R) symmetry during early embryogenesis in mouse, frog and zebrafish. The chick embryo, however, does not require motile cilia for L-R symmetry breaking. The diversity of mechanisms for L-R symmetry breaking among vertebrates and the trigger for such symmetry breaking in non-mammalian amniotes have remained unknown. Here we examined how L-R asymmetry is established in two reptiles, Madagascar ground gecko and Chinese softshell turtle. Both of these reptiles appear to lack motile cilia at the LRO. The expression of the Nodal gene at the LRO in the reptilian embryos was found to be asymmetric, in contrast to that in vertebrates such as mouse that are dependent on cilia for L-R patterning. Two paralogues of the Nodal gene derived from an ancient gene duplication are retained and expressed differentially in cilia-dependent and cilia-independent vertebrates. The expression of these two Nodal paralogues is similarly controlled in the lateral plate mesoderm but regulated differently at the LRO. Our in-depth analysis of reptilian embryos thus suggests that mammals and non-mammalian amniotes deploy distinct strategies dependent on different Nodal paralogues for rendering Nodal activity asymmetric at the LRO.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41559-019-1072-2DOI Listing
February 2020

Behavioral analysis in mice deficient for GAREM2 (Grb2-associated regulator of Erk/MAPK subtype2) that is a subtype of highly expressing in the brain.

Mol Brain 2019 11 12;12(1):94. Epub 2019 Nov 12.

The Faculty of Life and Environmental Sciences, Prefectural University of Hiroshima, 5562 Nanatsuka, Shobara, Hiroshima, 727-0023, Japan.

Grb2-associated regulator of Erk/MAPK (GAREM), is an adaptor protein related to the several cell growth factor receptor-signaling. The GAREM family has two subtypes, GAREM1 and GAREM2, both encoded in the human and mouse genome. Recent genome-wide research identified GAREM2 as a candidate of neurodegenerative diseases. Here, we use knockout (KO) mice to show the role of GAREM2, that is highly expressed in the brain. According to the comprehensive behavioral battery, they exhibited less anxiety both in elevated plus maze and open field tests, mildly increased social approaching behavior in the reciprocal social interaction test, and longer latency to immobility in the tail suspension test as compared to wild-type (WT). Additionally, the extension of neurites in the primary cultured neurons was suppressed in ones derived from GAREM2 KO mice. Furthermore, we also identified Intersectin, as a binding partner of GAREM2 in this study. Intersectin is also a multi-domain adaptor protein that regulates endocytosis and cell signaling, which can potentially alter the subcellular localization of GAREM2. The important molecules, such as the neurotrophin receptor and Erk family, that are involved in the signaling pathway of the neural cell growth in the mouse brain, have been reported to participate in emotional behavior. As GAREM plays a role in the cellular growth factor receptor signaling pathway, GAREM2 may have a common role related to the transduction of Erk signaling in the higher brain functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13041-019-0512-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6852768PMC
November 2019

Animal transgenesis now and beyond in the era of genome editing: Snapshots from the 15th Transgenic Technology Meeting (TT2019) in Kobe, Japan.

Genes Cells 2019 Dec 8;24(12):762-767. Epub 2019 Nov 8.

RIKEN Center for Biosystems Dynamics Research, Kobe, Japan.

The 15th Transgenic Technology (TT) Meeting of the International Society for Transgenic Technologies (ISTT) was held for the first time in Japan in April of 2019 (TT2019). Delegates from around the world, including researchers, technicians and trainees, spent an exciting 4 days, engaging in scientific and technical discussions, information sharing and networking. In the era of genome editing, CRISPR technologies prevailed as popular subjects of discussion throughout the meeting on topics ranging from their applications in the mouse and growing number of other research and industrial animal species, technical challenges in the production of genome-edited animals, to ethical considerations in biomedical research. In particular, impressive progress was reported on the use of CRISPR technologies in nonconventional animal models and large mammalian species in biomedical and industrial settings, indicating areas of expanding frontiers in animal transgenesis. Amid growing excitement with genome editing technologies, reports on conventional genetic engineering approaches and reproductive biology techniques demonstrated that these techniques remain essential to meet the demanding needs of generating complex genome modifications, complementing genome editing approaches. TT2019 was a great success, concluding with a widely shared appreciation of the current field and hints for future directions of animal transgenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/gtc.12725DOI Listing
December 2019

Identification of U11snRNA as an endogenous agonist of TLR7-mediated immune pathogenesis.

Proc Natl Acad Sci U S A 2019 11 6;116(47):23653-23661. Epub 2019 Nov 6.

Department of Molecular Immunology, Institute of Industrial Science, The University of Tokyo, 153-8505 Tokyo, Japan;

The activation of innate immune receptors by pathogen-associated molecular patterns (PAMPs) is central to host defense against infections. On the other hand, these receptors are also activated by immunogenic damage-associated molecular patterns (DAMPs), typically released from dying cells, and the activation can evoke chronic inflammatory or autoimmune disorders. One of the best known receptors involved in the immune pathogenesis is Toll-like receptor 7 (TLR7), which recognizes RNA with single-stranded structure. However, the causative DAMP RNA(s) in the pathogenesis has yet to be identified. Here, we first developed a chemical compound, termed KN69, that suppresses autoimmunity in several established mouse models. A subsequent search for KN69-binding partners led to the identification of U11 small nuclear RNA (U11snRNA) as a candidate DAMP RNA involved in TLR7-induced autoimmunity. We then showed that U11snRNA robustly activated the TLR7 pathway in vitro and induced arthritis disease in vivo. We also found a correlation between high serum level of U11snRNA and autoimmune diseases in human subjects and established mouse models. Finally, by revealing the structural basis for U11snRNA's ability to activate TLR7, we developed more potent TLR7 agonists and TLR7 antagonists, which may offer new therapeutic approaches for autoimmunity or other immune-driven diseases. Thus, our study has revealed a hitherto unknown immune function of U11snRNA, providing insight into TLR7-mediated autoimmunity and its potential for further therapeutic applications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1915326116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6876158PMC
November 2019

Prominin-1 Modulates Rho/ROCK-Mediated Membrane Morphology and Calcium-Dependent Intracellular Chloride Flux.

Sci Rep 2019 11 4;9(1):15911. Epub 2019 Nov 4.

Developmental Biomedical Science, Division of Biological Sciences, Nara Institute of Science and Technology, 8916-5, Takayama-cho, Ikoma, 630-0192, Japan.

Membrane morphology is an important structural determinant as it reflects cellular functions. The pentaspan membrane protein Prominin-1 (Prom1/CD133) is known to be localised to protrusions and plays a pivotal role in migration and the determination of cellular morphology; however, the underlying mechanism of its action have been elusive. Here, we performed molecular characterisation of Prom1, focussing primarily on its effects on cell morphology. Overexpression of Prom1 in RPE-1 cells triggers multiple, long, cholesterol-enriched fibres, independently of actin and microtubule polymerisation. A five amino acid stretch located at the carboxyl cytosolic region is essential for fibre formation. The small GTPase Rho and its downstream Rho-associated coiled-coil-containing protein kinase (ROCK) are also essential for this process, and active Rho colocalises with Prom1 at the site of initialisation of fibre formation. In mouse embryonic fibroblast (MEF) cells we show that Prom1 is required for chloride ion efflux induced by calcium ion uptake, and demonstrate that fibre formation is closely associated with chloride efflux activity. Collectively, these findings suggest that Prom1 affects cell morphology and contributes to chloride conductance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-52040-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6828804PMC
November 2019

A Novel Birthdate-Labeling Method Reveals Segregated Parallel Projections of Mitral and External Tufted Cells in the Main Olfactory System.

eNeuro 2019 Nov/Dec;6(6). Epub 2019 Nov 20.

Brain Function Laboratory, National Institute of Genetics.

A fundamental strategy in sensory coding is parallel processing, whereby unique, distinct features of sensation are computed and projected to the central target in the form of submodal maps. It remains unclear, however, whether such parallel processing strategy is employed in the main olfactory system, which codes the complex hierarchical odor and behavioral scenes. A potential scheme is that distinct subsets of projection neurons in the olfactory bulb (OB) form parallel projections to the targets. Taking advantage of the observation that the distinct projection neurons develop at different times, we developed a Cre-loxP-based method that allows for birthdate-specific labeling of cell bodies and their axon projections in mice. This birthdate tag analysis revealed that the mitral cells (MCs) born in an early developmental stage and the external tufted cells (TCs) born a few days later form segregated parallel projections. Specifically, the latter subset converges the axons onto only two small specific targets, one of which, located at the anterolateral edge of the olfactory tubercle (OT), excludes widespread MC projections. This target is made up of neurons that express dopamine D1 but not D2 receptor and corresponds to the most anterolateral isolation of the CAP compartments (aiCAP) that were defined previously. This finding of segregated projections suggests that olfactory sensing does indeed involve parallel processing of functionally distinct submodalities. Importantly, the birthdate tag method used here may pave the way for deciphering the functional meaning of these individual projection pathways in the future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/ENEURO.0234-19.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6868177PMC
June 2020

Metalloprotease-Dependent Attenuation of BMP Signaling Restricts Cardiac Neural Crest Cell Fate.

Cell Rep 2019 10;29(3):603-616.e5

Department of Regeneration Science and Engineering, Institute for Frontier Life and Medical Sciences, Kyoto University, Kyoto 606-8507, Japan. Electronic address:

In higher vertebrates, cephalic neural crest cells (NCCs) form craniofacial skeleton by differentiating into chondrocytes and osteoblasts. A subpopulation of cephalic NCCs, cardiac NCCs (CNCCs), migrates to the heart. However, CNCCs mostly do not yield skeletogenic derivatives, and the molecular mechanisms of this fate restriction remain elusive. We identify a disintegrin and metalloprotease 19 (Adam19) as a position-specific fate regulator of NCCs. Adam19-depleted mice abnormally form NCC-derived cartilage in their hearts through the upregulation of Sox9 levels in CNCCs. Moreover, NCC-lineage-specific Sox9-overexpressing mice recapitulate CNCC chondrogenesis. In vitro experiments show that Adam19 mediates the cleavage of bone morphogenic protein (BMP) type I receptor Alk2 (Acvr1), whereas pharmacogenetic approaches reveal that Adam19 inhibits CNCC chondrogenesis by suppressing the BMP-Sox9 cascade, presumably through processing Alk2. These findings suggest a metalloprotease-dependent mechanism attenuating cellular responsiveness to BMP ligands, which is essential for both the positional restriction of NCC skeletogenesis and normal heart development.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.09.019DOI Listing
October 2019

Modulation of autoimmune pathogenesis by T cell-triggered inflammatory cell death.

Nat Commun 2019 08 28;10(1):3878. Epub 2019 Aug 28.

Department of Molecular and Cellular Physiology, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501, Japan.

T cell-mediated autoimmunity encompasses diverse immunopathological outcomes; however, the mechanisms underlying this diversity are largely unknown. Dysfunction of the tripartite linear ubiquitin chain assembly complex (LUBAC) is associated with distinct autonomous immune-related diseases. Cpdm mice lacking Sharpin, an accessory subunit of LUBAC, have innate immune cell-predominant dermatitis triggered by death of LUBAC-compromised keratinocytes. Here we show that specific gene ablation of Sharpin in mouse Treg causes phenotypes mimicking cpdm-like inflammation. Mechanistic analyses find that multiple types of programmed cell death triggered by TNF from tissue-oriented T cells initiate proinflammatory responses to implicate innate immune-mediated pathogenesis in this T cell-mediated inflammation. Moreover, additional disruption of the Hoip locus encoding the catalytic subunit of LUBAC converts cpdm-like dermatitis to T cell-predominant autoimmune lesions; however, innate immune-mediated pathogenesis still remains. These findings show that T cell-mediated killing and sequential autoinflammation are common and crucial for pathogenic diversity during T cell-mediated autoimmune responses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-11858-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6713751PMC
August 2019

Induced 2C Expression and Implantation-Competent Blastocyst-like Cysts from Primed Pluripotent Stem Cells.

Stem Cell Reports 2019 09 8;13(3):485-498. Epub 2019 Aug 8.

Gladstone Institute of Cardiovascular Disease, San Francisco, CA 94158, USA; Department of Pharmacology, Faculty of Medicine, Osaka Medical College, Osaka 569-8686, Japan. Electronic address:

Soon after fertilization, the few totipotent cells of mammalian embryos diverge to form a structure called the blastocyst (BC). Although numerous cell types, including germ cells and extended-pluripotency stem cells, have been developed from pluripotent stem cells (PSCs) in vitro, generating functional BCs only from PSCs remains elusive. Here, we describe induced self-organizing 3D BC-like cysts (iBLCs) generated from mouse PSC culture. Resembling natural BCs, iBLCs have a blastocoel-like cavity and were formed with outer cells expressing trophectoderm lineage markers and with inner cells expressing pluripotency markers. iBLCs transplanted to pseudopregnant mice uteruses implanted, induced decidualization, and exhibited growth and development before resorption, demonstrating that iBLCs are implantation competent. iBLC precursor intermediates required the transcription factor Prdm14 and concomitantly activated the totipotency-related cleavage-stage MERVL reporter and 2C genes. Thus, our system may contribute to the understanding of molecular mechanisms underpinning totipotency, embryogenesis, and implantation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.stemcr.2019.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6739768PMC
September 2019

Dmrt factors determine the positional information of cerebral cortical progenitors via differential suppression of homeobox genes.

Development 2019 08 13;146(15). Epub 2019 Aug 13.

Laboratory for Cell Asymmetry, RIKEN Center for Biosystems Dynamics Research, Kobe, Hyogo 650-0047, Japan

The spatiotemporal identity of neural progenitors and the regional control of neurogenesis are essential for the development of cerebral cortical architecture. Here, we report that mammalian DM domain factors (Dmrt) determine the identity of cerebral cortical progenitors. Among the Dmrt family genes expressed in the developing dorsal telencephalon, and show a medial/lateral expression gradient. Their simultaneous loss confers a ventral identity to dorsal progenitors, resulting in the ectopic expression of Gsx2 and massive production of GABAergic olfactory bulb interneurons in the dorsal telencephalon. Furthermore, double-mutant progenitors in the medial region exhibit upregulated Pax6 and more lateral characteristics. These ventral and lateral shifts in progenitor identity depend on Dmrt gene dosage. We also found that Dmrt factors bind to and enhancers to suppress their expression. Our findings thus reveal that the graded expression of Dmrt factors provide positional information for progenitors by differentially repressing downstream genes in the developing cerebral cortex.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/dev.174243DOI Listing
August 2019

Translationally controlled tumor protein (TCTP) plays a pivotal role in cardiomyocyte survival through a Bnip3-dependent mechanism.

Cell Death Dis 2019 07 18;10(8):549. Epub 2019 Jul 18.

Cardiovascular Research Institute, Yokohama City University Graduate School of Medicine, Yokohama, Japan.

Prevention of cardiomyocyte death is an important therapeutic strategy for heart failure. In this study, we focused on translationally controlled tumor protein (TCTP), a highly conserved protein that is expressed ubiquitously in mammalian tissues, including heart. TCTP plays pivotal roles in survival of certain cell types, but its function in cardiomyocytes has not been examined. We aimed to clarify the role of TCTP in cardiomyocyte survival and the underlying mechanism. Here, we demonstrated that downregulation of TCTP with siRNA induced cell death of cardiomyocytes with apoptotic and autophagic features, accompanied with mitochondrial permeability transition pore (mPTP) opening. TCTP loss did not induce cell death of cardiac fibroblasts. Bcl-2/adenovirus E1B 19-kDa interacting protein 3 (Bnip3) was found to mediate the TCTP-loss-induced cardiomyocyte death. In exploring the clinical significance of the TCTP expression in the heart, we found that DOX treatment markedly downregulated the protein expression of TCTP in cultured cardiomyocytes and in mouse heart tissue. Exogenous rescue of TCTP expression attenuated DOX-induced cardiomyocyte death. In mice, cardiomyocyte-specific overexpression of TCTP resulted in decreased susceptibility to DOX-induced cardiac dysfunction, accompanied with attenuated induction of Bnip3. Dihydroartemisinin, a pharmacological TCTP inhibitor, induced development of heart failure and cardiomyocyte death in control mice, but not in mice with cardiomyocyte-specific TCTP overexpression. Our findings revealed TCTP has a pivotal role in cardiomyocyte survival, at least in part through a Bnip3-dependent mechanism. TCTP could be considered as a candidate therapeutic target to prevent DOX-induced heart failure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41419-019-1787-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639386PMC
July 2019

Author Correction: Sema7A/PlxnCl signaling triggers activity-dependent olfactory synapse formation.

Nat Commun 2019 Jul 10;10(1):3036. Epub 2019 Jul 10.

Department of Brain Function, University of Fukui School of Medicine, 23-3 Shimo-aizuki, Matsuoka, Fukui, 910-1193, Japan.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-11055-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620286PMC
July 2019

TRIOBP-5 sculpts stereocilia rootlets and stiffens supporting cells enabling hearing.

JCI Insight 2019 06 20;4(12). Epub 2019 Jun 20.

Department of Otolaryngology-Head and Neck Surgery, Kyoto University Graduate School of Medicine, Kyoto, Japan.

TRIOBP remodels the cytoskeleton by forming unusually dense F-actin bundles and is implicated in human cancer, schizophrenia, and deafness. Mutations ablating human and mouse TRIOBP-4 and TRIOBP-5 isoforms are associated with profound deafness, as inner ear mechanosensory hair cells degenerate after stereocilia rootlets fail to develop. However, the mechanisms regulating formation of stereocilia rootlets by each TRIOBP isoform remain unknown. Using 3 new Triobp mouse models, we report that TRIOBP-5 is essential for thickening bundles of F-actin in rootlets, establishing their mature dimensions and for stiffening supporting cells of the auditory sensory epithelium. The coiled-coil domains of this isoform are required for reinforcement and maintenance of stereocilia rootlets. A loss of TRIOBP-5 in mouse results in dysmorphic rootlets that are abnormally thin in the cuticular plate but have increased widths and lengths within stereocilia cores, and causes progressive deafness recapitulating the human phenotype. Our study extends the current understanding of TRIOBP isoform-specific functions necessary for life-long hearing, with implications for insight into other TRIOBPopathies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1172/jci.insight.128561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629139PMC
June 2019

Deletion of Class II ADP-Ribosylation Factors in Mice Causes Tremor by the Nav1.6 Loss in Cerebellar Purkinje Cell Axon Initial Segments.

J Neurosci 2019 08 14;39(32):6339-6353. Epub 2019 Jun 14.

Education and Research Support Center, Gunma University Graduate School of Medicine, Maebashi, Gunma 371-8511, Japan,

ADP-ribosylation factors (ARFs) are a family of small monomeric GTPases comprising six members categorized into three classes: class I (ARF1, 2, and 3), class II (ARF4 and 5), and class III (ARF6). In contrast to class I and III ARFs, which are the key regulators in vesicular membrane trafficking, the cellular function of class II ARFs remains unclear. In the present study, we generated class II ARF-deficient mice and found that ARF4/ARF5 mice exhibited essential tremor (ET)-like behaviors. electrophysiological recordings revealed that ARF4/ARF5 mice of both sexes exhibited abnormal brain activity when moving, raising the possibility of abnormal cerebellar excitability. Slice patch-clamp experiments demonstrated the reduced excitability of the cerebellar Purkinje cells (PCs) in ARF4/ARF5 mice. Immunohistochemical and electrophysiological analyses revealed a severe and selective decrease of pore-forming voltage-dependent Na channel subunit Nav1.6, important for maintaining repetitive action potential firing, in the axon initial segment (AIS) of PCs. Importantly, this decrease in Nav1.6 protein localized in the AIS and the consequent tremors in ARF4/ARF5 mice could be alleviated by the PC-specific expression of ARF5 using adeno-associated virus vectors. Together, our data demonstrate that the decreased expression of the class II ARF proteins in ARF4/ARF5 mice, leading to a haploinsufficiency of ARF4 in the absence of ARF5, impairs the localization of Nav1.6 to the AIS and hence reduces the membrane excitability in PCs, resulting in the ET-like movement disorder. We suggest that class II ARFs function in localizing specific proteins, such as Nav1.6, to the AIS. We found that decreasing the expression of class II ARF proteins, through the generation of ARF4/ARF5 mice, impairs Nav1.6 distribution to the axon initial segment (AIS) of cerebellar Purkinje cells (PCs), thereby resulting in the impairment of action potential firing of PCs. The ARF4/ARF5 mutant mice exhibited movement-associated essential tremor (ET)-like behavior with pharmacological profiles similar to those in ET patients. The exogenous expression of ARF5 reduced the tremor phenotype and restored the localization of Nav1.6 immunoreactivity to the AIS in ARF4/ARF5 mice. Thus, our results suggest that class II ARFs are involved in the localization of Nav1.6 to the AISs in cerebellar PCs and that the reduction of class II ARF activity leads to ET-like movement disorder.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1523/JNEUROSCI.2002-18.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6687904PMC
August 2019

Behavioral and neurological analyses of adult mice carrying null and distinct loss-of-receptor function mutations in protein tyrosine phosphatase receptor type Z (PTPRZ).

PLoS One 2019 13;14(6):e0217880. Epub 2019 Jun 13.

Division of Molecular Neurobiology, National Institute for Basic Biology (NIBB), Higashiyama, Myodaiji-cho, Okazaki, Aichi, Japan.

Protein tyrosine phosphatase receptor type Z (PTPRZ) is preferentially expressed in the central nervous system as two transmembrane receptor isoforms PTPRZ-A/B and one secretory isoform PTPRZ-S. Ptprz-knockout mice lacking the expression of all three isoforms show behavioral, learning, and neurological abnormalities, including increased exploratory activities to novelty, deficits in spatial and contextual learning, and reduced responses to methamphetamine, relative to wild-type mice. To investigate whether PTPRZ isoforms play distinct physiological roles, we herein performed behavioral studies on two knock-in mouse lines: One expresses the catalytically inactive Cys-1930 to Ser (CS) mutants of PTPRZ-A/B, while the other generated in the present study expresses catalytically active mutants of PTPRZ-A/B lacking the negative regulatory PTP-D2 domain and C-terminal PDZ-binding motif (ΔD2) instead of wild-type PTPRZ-A/-B. In contrast to Ptprz-knockout mice, neither increased responses to novelty in the open field nor memory impairments in the inhibitory-avoidance task were observed in Ptprz-CS or Ptprz-ΔD2 mice. However, the effects of methamphetamine on locomotor activity were significantly weaker in Ptprz-KO mice and CS mutant mice than in wild-type mice, but were normal in ΔD2 mutant mice. Furthermore, microdialysis experiments revealed that methamphetamine-evoked dopamine release in the nucleus accumbens was reduced in Ptprz-KO mice and CS mutant mice. These results suggest that the extracellular region of PTPRZ, including the secretory isoform, is crucial for behavioral responses to novelty and the formation of aversive memories, whereas the PTPase activities of PTPRZ receptor isoforms are involved in regulating the dopaminergic system.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0217880PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6563982PMC
February 2020

Non-coding cis-element of Period2 is essential for maintaining organismal circadian behaviour and body temperature rhythmicity.

Nat Commun 2019 06 12;10(1):2563. Epub 2019 Jun 12.

Department of Systems Biology, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyō-ku, Kyoto, 606-8501, Japan.

Non-coding cis-regulatory elements are essential determinants of development, but their exact impacts on behavior and physiology in adults remain elusive. Cis-element-based transcriptional regulation is believed to be crucial for generating circadian rhythms in behavior and physiology. However, genetic evidence supporting this model is based on mutations in the protein-coding sequences of clock genes. Here, we report generation of mutant mice carrying a mutation only at the E'-box cis-element in the promoter region of the core clock gene Per2. The Per2 E'-box mutation abolishes sustainable molecular clock oscillations and renders circadian locomotor activity and body temperature rhythms unstable. Without the E'-box, Per2 messenger RNA and protein expression remain at mid-to-high levels. Our work delineates the Per2 E'-box as a critical nodal element for keeping sustainable cell-autonomous circadian oscillation and reveals the extent of the impact of the non-coding cis-element in daily maintenance of animal locomotor activity and body temperature rhythmicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-10532-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6561950PMC
June 2019

Structured spike series specify gene expression patterns for olfactory circuit formation.

Science 2019 07 6;365(6448). Epub 2019 Jun 6.

Laboratory of Chemical Pharmacology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan.

Neural circuits emerge through the interplay of genetic programming and activity-dependent processes. During the development of the mouse olfactory map, axons segregate into distinct glomeruli in an olfactory receptor (OR)-dependent manner. ORs generate a combinatorial code of axon-sorting molecules whose expression is regulated by neural activity. However, it remains unclear how neural activity induces OR-specific expression patterns of axon-sorting molecules. We found that the temporal patterns of spontaneous neuronal spikes were not spatially organized but were correlated with the OR types. Receptor substitution experiments demonstrated that ORs determine spontaneous activity patterns. Moreover, optogenetically differentiated patterns of neuronal activity induced specific expression of the corresponding axon-sorting molecules and regulated axonal segregation. Thus, OR-dependent temporal patterns of spontaneous activity play instructive roles in generating the combinatorial code of axon-sorting molecules during olfactory map formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1126/science.aaw5030DOI Listing
July 2019

Gli signaling pathway modulates fibroblast activation and facilitates scar formation in pulmonary fibrosis.

Biochem Biophys Res Commun 2019 06 8;514(3):684-690. Epub 2019 May 8.

Department of Molecular Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan; Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, Noda, Japan; Japan Agency for Medical Research and Development-CREST Program, Tokyo, Japan. Electronic address:

Pulmonary fibrosis is characterized by progressive and irreversible scarring of alveoli, which causes reduction of surface epithelial area and eventually respiratory failure. The precise mechanism of alveolar scarring is poorly understood. In this study, we explored transcriptional signatures of activated fibroblasts in alveolar airspaces by using intratracheal transfer in bleomycin-induced lung fibrosis. Lung fibroblasts transferred into injured alveoli upregulated genes related to translation and metabolism in the first two days, and upregulated genes related to extracellular matrix (ECM) production between day 2 and 7. Upstream analysis of these upregulated genes suggested possible contribution of hypoxia-inducible factors 1a (Hif1a) to fibroblast activation in the first two days, and possible contribution of kruppel-like factor 4 (Klf4) and glioma-associated oncogene (Gli) transcription factors to fibroblast activation in the following profibrotic phase. Fibroblasts purified based on high Acta2 expression after intratracheal transfer were also characterized by ECM production and upstream regulation by Klf4 and Gli proteins. Pharmacological inhibition of Gli proteins by GANT61 in bleomycin-induced lung fibrosis altered the pattern of scarring characterized by dilated airspaces and smaller fibroblast clusters. Activated fibroblasts isolated from GANT61-treated mice showed decreased migration capacity, suggesting that Gli signaling inhibition attenuated fibroblast activation. In conclusion, we revealed transcriptional signatures and possible upstream regulators of activated fibroblasts in injured alveolar airspaces. The altered scar formation by Gli signaling inhibition supports that activated fibroblasts in alveolar airspaces may play a critical role in scar formation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrc.2019.05.011DOI Listing
June 2019

Asrij/OCIAD1 suppresses CSN5-mediated p53 degradation and maintains mouse hematopoietic stem cell quiescence.

Blood 2019 05 5;133(22):2385-2400. Epub 2019 Apr 5.

Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore, India.

Inactivation of the tumor suppressor p53 is essential for unrestrained growth of cancers. However, only 11% of hematological malignancies have mutant p53. Mechanisms that cause wild-type p53 dysfunction and promote leukemia are inadequately deciphered. The stem cell protein Asrij/OCIAD1 is misexpressed in several human hematological malignancies and implicated in the p53 pathway and DNA damage response. However, Asrij function in vertebrate hematopoiesis remains unknown. We generated the first null (knockout [KO]) mice and show that they are viable and fertile with no gross abnormalities. However, by 6 months, they exhibit increased peripheral blood cell counts, splenomegaly, and an expansion of bone marrow hematopoietic stem cells (HSCs) with higher myeloid output. HSCs lacking Asrij are less quiescent and more proliferative with higher repopulation potential as observed from serial transplantation studies. However, stressing KO mice with sublethal γ irradiation or multiple injections of 5-fluorouracil results in reduced survival and rapid depletion of hematopoietic stem/progenitor cells (HSPCs) by driving them into proliferative exhaustion. Molecular and biochemical analyses revealed increased polyubiquitinated protein levels, Akt/STAT5 activation and COP9 signalosome subunit 5 (CSN5)-mediated p53 ubiquitination, and degradation in KO HSPCs. Further, we show that Asrij sequesters CSN5 via its conserved OCIA domain, thereby preventing p53 degradation. In agreement, Nutlin-3 treatment of KO mice restored p53 levels and reduced high HSPC frequencies. Thus, we provide a new mouse model resembling myeloproliferative disease and identify a posttranslational regulator of wild-type p53 essential for maintaining HSC quiescence that could be a potential target for pharmacological intervention.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2019000530DOI Listing
May 2019