Publications by authors named "Nobuaki Yoshida"

77 Publications

Generation of a p16 Reporter Mouse and Its Use to Characterize and Target p16 Cells In Vivo.

Cell Metab 2020 11 18;32(5):814-828.e6. Epub 2020 Sep 18.

Division of Molecular Regulation of Inflammatory and Immune Diseases, Research Institute of Biomedical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-0022, Japan.

Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-Cre-tdTomato mouse model to analyze the in vivo characteristics of p16 cells at a single-cell level. We found tdTomato-positive p16 cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16 cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16 cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.
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http://dx.doi.org/10.1016/j.cmet.2020.09.006DOI Listing
November 2020

Impact of spaceflight on the murine thymus and mitigation by exposure to artificial gravity during spaceflight.

Sci Rep 2019 12 27;9(1):19866. Epub 2019 Dec 27.

Laboratory for Immune Homeostasis, RIKEN Center for Integrative Medical Sciences, Yokohama, 230-0045, Japan.

The environment experienced during spaceflight may impact the immune system and the thymus appears to undergo atrophy during spaceflight. However, molecular aspects of this thymic atrophy remain to be elucidated. In this study, we analysed the thymi of mice on board the international space station (ISS) for approximately 1 month. Thymic size was significantly reduced after spaceflight. Notably, exposure of mice to 1 × g using centrifugation cages in the ISS significantly mitigated the reduction in thymic size. Although spaceflight caused thymic atrophy, the global thymic structure was not largely changed. However, RNA sequencing analysis of the thymus showed significantly reduced expression of cell cycle-regulating genes in two independent spaceflight samples. These reductions were partially countered by 1 × g exposure during the space flights. Thus, our data suggest that spaceflight leads to reduced proliferation of thymic cells, thereby reducing the size of the thymus, and exposure to 1 × g might alleviate the impairment of thymus homeostasis induced by spaceflight.
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http://dx.doi.org/10.1038/s41598-019-56432-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934594PMC
December 2019

Down-regulation of GATA1-dependent erythrocyte-related genes in the spleens of mice exposed to a space travel.

Sci Rep 2019 05 21;9(1):7654. Epub 2019 May 21.

RIKEN Center for Integrative Medical Sciences, Yokohama, 230-0045, Japan.

Secondary lymphoid organs are critical for regulating acquired immune responses. The aim of this study was to characterize the impact of spaceflight on secondary lymphoid organs at the molecular level. We analysed the spleens and lymph nodes from mice flown aboard the International Space Station (ISS) in orbit for 35 days, as part of a Japan Aerospace Exploration Agency mission. During flight, half of the mice were exposed to 1 g by centrifuging in the ISS, to provide information regarding the effect of microgravity and 1 g exposure during spaceflight. Whole-transcript cDNA sequencing (RNA-Seq) analysis of the spleen suggested that erythrocyte-related genes regulated by the transcription factor GATA1 were significantly down-regulated in ISS-flown vs. ground control mice. GATA1 and Tal1 (regulators of erythropoiesis) mRNA expression was consistently reduced by approximately half. These reductions were not completely alleviated by 1 g exposure in the ISS, suggesting that the combined effect of space environments aside from microgravity could down-regulate gene expression in the spleen. Additionally, plasma immunoglobulin concentrations were slightly altered in ISS-flown mice. Overall, our data suggest that spaceflight might disturb the homeostatic gene expression of the spleen through a combination of microgravity and other environmental changes.
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http://dx.doi.org/10.1038/s41598-019-44067-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6529412PMC
May 2019

Foxr2 promotes formation of CNS-embryonal tumors in a Trp53-deficient background.

Neuro Oncol 2019 08;21(8):993-1004

Division of Molecular and Developmental Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Background: Embryonal tumors in the central nervous system (CNS) are primary, aggressive, and poorly differentiated pediatric brain tumors. We identified forkhead box R2 (Foxr2) as an oncogene for medulloblastoma through a transposon-based insertional mutagenesis screen. Foxr2 translocation has been identified in a subset of human embryonal tumors of the CNS, designated as CNS neuroblastoma with Foxr2 activation (CNS NB-Foxr2); however, the in vivo functions of Foxr2 remain elusive.

Methods: We analyzed the effect of Foxr2 overexpression in the mouse brain by generating a transgenic strain that expresses Foxr2 in the entire brain under a transformation related protein 53 (Trp53)-deficient background. We performed histological analysis of tumors and characterized tumor-derived sphere-forming cells. We investigated gene expression profiles of tumor-derived cells.

Results: Foxr2 and Trp53 loss promoted tumor formation in the olfactory bulb (OB) and brainstem (BS). The tumors showed the common morphological features of small round blue cell tumors, exhibiting divergent, mainly neuronal and glial, patterns of differentiation, which corresponds to the definition of CNS-embryonal tumors. Importantly, all mice developed CNS-embryonal tumors. In the OB, early proliferative lesions consisting of oligodendrocyte transcription factor 2 (Olig2+) cells were observed, indicating that Foxr2 expression expanded Olig2+ cells in the OB. Tumor-derived cells formed spheres in vitro and induced tumors that recapitulated the parental tumor upon transplantation, indicating the presence of tumor-initiating cells. Gene expression profiling revealed that OB and BS tumor cells were enriched for the expression of the genes specific to CNS NB-Foxr2.

Conclusion: Our data demonstrate that Foxr2 plays a causative role in the formation of CNS-embryonal tumors.
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http://dx.doi.org/10.1093/neuonc/noz067DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6682215PMC
August 2019

CBP/p300 antagonises EGFR-Ras-Erk signalling and suppresses increased Ras-Erk signalling-induced tumour formation in mice.

J Pathol 2019 09 3;249(1):39-51. Epub 2019 May 3.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

CREB-binding protein (CBP) and p300 have oncogenic properties; both co-operate with pro-oncogenic transcription factors downstream of Ras-Erk signalling to support cell proliferation. By contrast, missense, truncating and in-frame mutations of CBP/p300 are found frequently in some human cancers, including cutaneous squamous cell carcinomas that originate from epidermal keratinocytes. Data support that dysfunction of CBP/p300 contributes to keratinocyte hyperproliferation and tumourigenesis; however, the mechanism by which dysfunction of CBP/p300 affects keratinocytes is unknown. Here, we used mice harbouring keratinocyte-specific genetic modifications to examine the role of CBP/p300 in the epidermis. While a single copy of either Crebbp or Ep300 was necessary and sufficient for maintaining epidermal development, reduced expression of CBP/p300 strengthened the Ras-Erk signalling-induced hyperplastic phenotype of epidermal keratinocytes. Reduced CBP/p300 expression increased ligand-induced EGFR activity while decreasing basal expression of Mig6, a negative regulator of EGFR. A reduction in CBP/p300, in combination with increased Ras-Erk signalling, also promoted epidermal tumour formation in mice. Thus, our findings support that CBP/p300 acts as a tumour suppressor in epidermal keratinocytes by counteracting EGFR-Ras-Erk signalling. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.5279DOI Listing
September 2019

RNA-binding protein Ptbp1 is essential for BCR-mediated antibody production.

Int Immunol 2019 03;31(3):157-166

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Shirokanedai, Minato-ku, Tokyo, Japan.

The RNA-binding protein polypyrimidine tract-binding protein-1 (Ptbp1) binds to the pyrimidine-rich sequence of target RNA and controls gene expression via post-transcriptional regulation such as alternative splicing. Although Ptbp1 is highly expressed in B lymphocytes, its role to date is largely unknown. To clarify the role of Ptbp1 in B-cell development and function, we generated B-cell-specific Ptbp1-deficient (P1BKO) mice. B-cell development in the bone marrow, spleen and peritoneal cavity of the P1BKO mice was nearly normal. However, the P1BKO mice had significantly lower levels of natural antibodies in serum compared with those of the control mice. To investigate the effect of Ptbp1 deficiency on the immune response in vivo, we immunized the P1BKO mice with T-cell-independent type-2 (TI-2) antigen NP-Ficoll and T-cell-dependent (TD) antigen NP-CGG. We found that B-cell-specific Ptbp1 deficiency causes an immunodeficiency phenotype due to defective production of antibody against both TI-2 and TD antigen. This immunodeficiency was accompanied by impaired B-cell receptor (BCR)-mediated B-cell activation and plasmablast generation. These findings demonstrate that Ptbp1 is essential for the humoral immune response.
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http://dx.doi.org/10.1093/intimm/dxy077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6400050PMC
March 2019

PTBP1 contributes to spermatogenesis through regulation of proliferation in spermatogonia.

J Reprod Dev 2019 Feb 12;65(1):37-46. Epub 2018 Nov 12.

Laboratory of Reproductive Systems Biology, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo 108-8639, Japan.

Polypyrimidine tract-binding protein 1 (PTBP1) is a highly conserved RNA-binding protein that is a well-known regulator of alternative splicing. Testicular tissue is one of the richest tissues with respect to the number of alternative splicing mRNA isoforms, but the molecular role(s) of PTBP1 in the regulation of these isoforms during spermatogenesis is still unclear. Here, we developed a germ cell-specific Ptbp1 conditional knockout (cKO) mouse model by using the Cre-loxP system to investigate the role of PTBP1 in spermatogenesis. Testis weight in Ptbp1 cKO mice was comparable to that in age-matched controls until 3 weeks of age; at ≥ 2 months old, testis weight was significantly lighter in cKO mice than in age-matched controls. Sperm count in Ptbp1 cKO mice at 2 months old was comparable to that in controls, whereas sperm count significantly decreased at 6 months old. Seminiferous tubules that exhibited degeneration in spermatogenic function were more evident in the 2-month-old Ptbp1 cKO mice than in controls. In addition, the early neonatal proliferation of spermatogonia, during postnatal days 1-5, was significantly retarded in Ptbp1 cKO mice compared with that in controls. An in vitro spermatogonia culture model (germline stem cells) revealed that hydroxytamoxifen-induced deletion of PTBP1 from germline stem cells caused severe proliferation arrest accompanied by an increase of apoptotic cell death. These data suggest that PTBP1 contributes to spermatogenesis through regulation of spermatogonia proliferation.
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http://dx.doi.org/10.1262/jrd.2018-109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6379764PMC
February 2019

Cleavage of Toll-Like Receptor 9 Ectodomain Is Required for Responses to Single Strand DNA.

Front Immunol 2018 27;9:1491. Epub 2018 Jun 27.

Division of Innate Immunity, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Mouse toll-like receptor 9 (TLR9) is an endosomal sensor for single-stranded DNA. TLR9 is transported from the endoplasmic reticulum to endolysosomes by a multiple transmembrane protein Unc93 homolog B1, and proteolytically cleaved at its ectodomain. The structure of TLR9 and its biochemical analyses have shown that the proteolytic cleavage of TLR9 ectodomain enables TLR9-dimerization and TLR9 activation. However, the requirement of TLR9 cleavage has not been studied. We here show that the 13 amino acids deletion at the cleavage site made TLR9 resistant to proteolytic cleavage. The deletion mutation in the gene impaired TLR9-dependent cytokine production in conventional dendritic cells from the mutant mice. Not only production of inflammatory cytokines (TNF-α and IL-12p40), chemokine (CCR5/RANTES), and type I interferon (IFN-α) induced by administration of TLR9 ligand was also impaired. These results demonstrate that the TLR9 cleavage is required for TLR9 responses .
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http://dx.doi.org/10.3389/fimmu.2018.01491DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6030252PMC
June 2018

Long-term hindlimb unloading causes a preferential reduction of medullary thymic epithelial cells expressing autoimmune regulator (Aire).

Biochem Biophys Res Commun 2018 06;501(3):745-750

Center for Integrative Medical Science, RIKEN, Yokohama 230-0045, Japan; Mouse Epigenetics Project, ISS/Kibo experiment, Japan Aerospace Exploration Agency (JAXA), Tsukuba, Japan. Electronic address:

Hindlimb unloading (HU) of rodents has been used as a ground-based model of spaceflight. In this study, we investigated the detailed impact of 14-day HU on the murine thymus. Thymic mass and cell number were significantly reduced after 14 days of hindlimb unloading, which was accompanied by an increment of plasma corticosterone. Although corticosterone reportedly causes selective apoptosis of CD4CD8 thymocytes (CD4CD8DPs) in mice treated with short-term HU, the reduction of thymocyte cellularity after the 14-day HU was not selective for CD4CD8DPs. In addition to the thymocyte reduction, the cellularity of thymic epithelial cells (TECs) was also reduced by the 14-day HU. Flow cytometric and RNA-sequencing analysis suggested that medullary TECs (mTECs) were preferentially reduced after HU. Moreover, immunohistochemical staining suggested that the 14-day HU caused a reduction of the mTECs expressing autoimmune regulator (Aire). Our data suggested that HU impacts both thymocytes and TECs. Consequently, these data imply that thymic T cell repertoire formation could be disturbed during spaceflight-like stress.
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http://dx.doi.org/10.1016/j.bbrc.2018.05.060DOI Listing
June 2018

Foxc2 knock-in mice mark stage-specific Foxc2-expressing cells during mouse organogenesis.

Congenit Anom (Kyoto) 2017 Jan;57(1):24-31

Department of Biochemistry, Hamamatsu University School of Medicine, Hamamatsu, Japan.

Foxc2, a member of the winged helix transcription factor family, is essential for eye, calvarial bone, cardiovascular and kidney development in mice. Nevertheless, how Foxc2-expressing cells and their descendent cells contribute to the development of these tissues and organs has not been elucidated. Here, we generated a Foxc2 knock-in (Foxc2 ) mouse, in which administration of estrogen receptor antagonist tamoxifen induces nuclear translocation of Cre recombinase in Foxc2-expressing cells. By crossing with ROSA-LacZ reporter mice (Foxc2 ; R26R), the fate of Foxc2 positive (Foxc2 ) cells was analyzed through LacZ staining at various embryonic stages. We found Foxc2 cell descendants in the supraoccipital and exoccipital bone in E18.5 embryos, when tamoxifen was administered at embryonic day (E) 8.5. Furthermore, Foxc2 descendant cranial neural crest cells at E8-10 were restricted to the corneal mesenchyme, while Foxc2 cell derived cardiac neural crest cells at E6-12 were found in the aorta, pulmonary trunk and valves, and endocardial cushions. Foxc2 cell descendant contributions to the glomerular podocytes in the kidney were also observed following E6.5 tamoxifen treatment. Our results are consistent with previous reports of Foxc2 expression during early embryogenesis and the Foxc2 mouse provides a tool to investigate spatiotemporal roles of Foxc2 and contributions of Foxc2 expressing cells during mouse embryogenesis.
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http://dx.doi.org/10.1111/cga.12198DOI Listing
January 2017

Role of transcription factor Tgif2 in photoreceptor differentiation in the mouse retina.

Exp Eye Res 2016 11 14;152:34-42. Epub 2016 Sep 14.

Division of Molecular and Developmental Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan. Electronic address:

5'TG3'-interacting factors (TGIFs) function as transcriptional repressors. Defects in TGIFs cause severe abnormalities in the developing brain and face. We found that Tgif2 was highly expressed in the mouse retina at early stages of development and examined its role in retinal development. Knockdown of Tgif2 in retinal explants at E14 using shRNA (sh-Tgif2) resulted in a decreased number of rod photoreceptors, whereas the number of cone photoreceptors increased without perturbation of cell proliferation and apoptosis. Concomitantly, the expression levels of photoreceptor-related genes were decreased in sh-Tgif2-introduced retinal explants. To examine the in vivo effects of Tgif2 overexpression, we generated Tgif2 conditional knock-in mice (Tgif2cKI). Although retinal cell differentiation, based on the relative proportions of retinal subtypes in the mature retina, was not affected, we observed abnormal localization of cone photoreceptor cell nuclei in the outer nuclear layer of the Tgif2cKI retina. However, electrical retinography suggest that cones in Tgif2cKI were functionally equivalent to those of wild mice. Our study revealed that Tgif2 participates in photoreceptor cell differentiation in the early stages of retinal development and regulates proper subretinal localization of the cone photoreceptors.
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http://dx.doi.org/10.1016/j.exer.2016.09.005DOI Listing
November 2016

Cell-Type-Specific Alternative Splicing Governs Cell Fate in the Developing Cerebral Cortex.

Cell 2016 Aug;166(5):1147-1162.e15

Division of Genetics and Genomics, Manton Center for Orphan Disease Research, Howard Hughes Medical Institute, Boston Children's Hospital, Boston, MA 02115, USA; Departments of Neurology and Pediatrics, Harvard Medical School, Boston, MA 02115, USA; Harvard Stem Cell Institute, Cambridge, MA 02138, USA; Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA. Electronic address:

Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development.
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http://dx.doi.org/10.1016/j.cell.2016.07.025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5248659PMC
August 2016

Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator.

J Exp Med 2016 07 11;213(8):1441-58. Epub 2016 Jul 11.

Division of Cellular and Molecular Biology, The Institute of Medical Science, The University of Tokyo, Shirokane-dai, Minato-ku, Tokyo 108-8639, Japan

Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire(+) mTECs) is unclear. Here, we describe novel embryonic precursors of Aire(+) mTECs. We found the candidate precursors of Aire(+) mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire(+) mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire(+) mTECs and efficiently suppressed the onset of autoimmunity induced by Aire(+) mTEC deficiency. Mechanistically, pMECs differentiated into Aire(+) mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire(+) mTECs.
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http://dx.doi.org/10.1084/jem.20151780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4986530PMC
July 2016

The Histone Demethylase FBXL10 Regulates the Proliferation of Spermatogonia and Ensures Long-Term Sustainable Spermatogenesis in Mice.

Biol Reprod 2016 Apr 16;94(4):92. Epub 2016 Mar 16.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan

The F-box and leucine-rich repeat protein 10 (Fbxl10) gene encodes a protein that catalyzes demethylation of H3K4 and H3K36. In this study, we show the important roles of FBXL10 as a histone demethylase in sustainable sperm production using mice in which the JmjC domain of Fbxl10 was deleted (Fbxl10(DeltaJ/DeltaJ)). In histological analysis, testis sections from 10-wk-old Fbxl10(DeltaJ/DeltaJ) mice appeared normal. On the other hand, testes from 7-mo-old Fbxl10(DeltaJ/DeltaJ) mice contained a greater ratio of seminiferous tubules exhibiting degeneration of spermatogenesis. Further analysis using an in vitro spermatogonia culture system, that is, germline stem cells (GSCs), revealed that Fbxl10(DeltaJ/DeltaJ) GSCs expressed a significantly higher level of P21 and P19 mRNA, cyclin-dependent kinase inhibitors and also known as cellular senescence markers, than wild-type (WT) GSCs. Furthermore, the ratio of Fbxl10(DeltaJ/DeltaJ) GSCs in G0/G1 phase was higher and the ratios in S and G2/M phases were lower than the corresponding ratios of WT GSCs, and the doubling speed of Fbxl10(DeltaJ/DeltaJ) GSCs was significantly slower than that of WT GSCs. In addition to these in vitro results, an in vivo study indicated that recovery of spermatogenesis after a transient reduction in the number of testicular germ cells by busulfan treatment was significantly slower in Fbxl10(DeltaJ/DeltaJ) mice than in WT mice. These data suggest that Fbxl10 plays important roles in long-term sustainable spermatogenesis via regulating cell cycle.
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http://dx.doi.org/10.1095/biolreprod.115.135988DOI Listing
April 2016

Phospholipase Cγ2 Is Required for Luminal Expansion of the Epididymal Duct during Postnatal Development in Mice.

PLoS One 2016 7;11(3):e0150521. Epub 2016 Mar 7.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo, Japan.

Phospholipase Cγ2 (PLCγ2)-deficient mice exhibit misconnections of blood and lymphatic vessels, and male infertility. However, the cell type responsible for vascular partitioning and the mechanism for male infertility remain unknown. Accordingly, we generated a mouse line that conditionally expresses endogenous Plcg2 in a Cre/loxP recombination-dependent manner, and found that Tie2-Cre- or Pf4-Cre-driven reactivation of Plcg2 rescues PLCγ2-deficient mice from the vascular phenotype. By contrast, male mice rescued from the vascular phenotype exhibited epididymal sperm granulomas. As judged from immunostaining, PLCγ2 was expressed in clear cells in the epididymis. PLCγ2 deficiency did not compromise differentiation of epididymal epithelial cells, including clear cells, and tube formation at postnatal week 2. However, luminal expansion of the epididymal duct was impaired during the prepubertal period, regardless of epithelial cell polarity and tube architecture. These results suggest that PLCγ2-deficient clear cells cause impaired luminal expansion, stenosis of the epididymal duct, attenuation of luminal flow, and subsequent sperm granulomas. Clear cell-mediated luminal expansion is also supported by the observation that PLCγ2-deficient males were rescued from infertility by epididymal epithelium-specific reactivation of Plcg2, although the edematous and hemorrhagic phenotype associated with PLCγ2 deficiency also caused spontaneous epididymal sperm granulomas in aging males. Collectively, our findings demonstrate that PLCγ2 in clear cells plays an essential role in luminal expansion of the epididymis during the prepubertal period in mice, and reveal an unexpected link between PLCγ2, clear cells, and epididymal development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0150521PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4780702PMC
August 2016

Establishment of a tamoxifen-inducible Cre-driver mouse strain for widespread and temporal genetic modification in adult mice.

Exp Anim 2016 Jul 29;65(3):231-44. Epub 2016 Feb 29.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Temporal genetic modification of mice using the ligand-inducible Cre/loxP system is an important technique that allows the bypass of embryonic lethal phenotypes and access to adult phenotypes. In this study, we generated a tamoxifen-inducible Cre-driver mouse strain for the purpose of widespread and temporal Cre recombination. The new line, named CM32, expresses the GFPneo-fusion gene in a wide variety of tissues before FLP recombination and tamoxifen-inducible Cre after FLP recombination. Using FLP-recombined CM32 mice (CM32Δ mice) and Cre reporter mouse lines, we evaluated the efficiency of Cre recombination with and without tamoxifen administration to adult mice, and found tamoxifen-dependent induction of Cre recombination in a variety of adult tissues. In addition, we demonstrated that conditional activation of an oncogene could be achieved in adults using CM32Δ mice. CM32Δ;T26 mice, which harbored a Cre recombination-driven, SV40 large T antigen-expressing transgene, were viable and fertile. No overt phenotype was found in the mice up to 3 months after birth. Although they displayed pineoblastomas (pinealoblastomas) and/or thymic enlargement due to background Cre recombination by 6 months after birth, they developed epidermal hyperplasia when administered tamoxifen. Collectively, our results suggest that the CM32Δ transgenic mouse line can be applied to the assessment of adult phenotypes in mice with loxP-flanked transgenes.
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http://dx.doi.org/10.1538/expanim.15-0126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4976237PMC
July 2016

Ground-based assessment of JAXA mouse habitat cage unit by mouse phenotypic studies.

Exp Anim 2016 May 28;65(2):175-87. Epub 2016 Jan 28.

Mouse Epigenetics Project, ISS/Kibo experiment, JAXA, Japan.

The Japan Aerospace Exploration Agency developed the mouse Habitat Cage Unit (HCU) for installation in the Cell Biology Experiment Facility (CBEF) onboard the Japanese Experimental Module ("Kibo") on the International Space Station. The CBEF provides "space-based controls" by generating artificial gravity in the HCU through a centrifuge, enabling a comparison of the biological consequences of microgravity and artificial gravity of 1 g on mice housed in space. Therefore, prior to the space experiment, a ground-based study to validate the habitability of the HCU is necessary to conduct space experiments using the HCU in the CBEF. Here, we investigated the ground-based effect of a 32-day housing period in the HCU breadboard model on male mice in comparison with the control cage mice. Morphology of skeletal muscle, the thymus, heart, and kidney, and the sperm function showed no critical abnormalities between the control mice and HCU mice. Slight but significant changes caused by the HCU itself were observed, including decreased body weight, increased weights of the thymus and gastrocnemius, reduced thickness of cortical bone of the femur, and several gene expressions from 11 tissues. Results suggest that the HCU provides acceptable conditions for mouse phenotypic analysis using CBEF in space, as long as its characteristic features are considered. Thus, the HCU is a feasible device for future space experiments.
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http://dx.doi.org/10.1538/expanim.15-0077DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4873486PMC
May 2016

Hypergravity Provokes a Temporary Reduction in CD4+CD8+ Thymocyte Number and a Persistent Decrease in Medullary Thymic Epithelial Cell Frequency in Mice.

PLoS One 2015 29;10(10):e0141650. Epub 2015 Oct 29.

Division of Cellular and Molecular Biology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Mouse epigenetics project, ISS/Kibo experiment, Japan Aerospace Exploration Agency, JAXA, Tsukuba, Japan.

Gravity change affects many immunological systems. We investigated the effects of hypergravity (2G) on murine thymic cells. Exposure of mice to 2G for three days reduced the frequency of CD4+CD8+ thymocytes (DP) and mature medullary thymic epithelial cells (mTECs), accompanied by an increment of keratin-5 and keratin-8 double-positive (K5+K8+) TECs that reportedly contain TEC progenitors. Whereas the reduction of DP was recovered by a 14-day exposure to 2G, the reduction of mature mTECs and the increment of K5+K8+ TEC persisted. Interestingly, a surgical lesion of the inner ear's vestibular apparatus inhibited these hypergravity effects. Quantitative PCR analysis revealed that the gene expression of Aire and RANK that are critical for mTEC function and development were up-regulated by the 3-day exposure and subsequently down-regulated by the 14-day exposure to 2G. Unexpectedly, this dynamic change in mTEC gene expression was independent of the vestibular apparatus. Overall, data suggest that 2G causes a temporary reduction of DP and a persistent reduction of mature mTECs in a vestibular system-dependent manner, and also dysregulates mTEC gene expression without involving the vestibular system. These data might provide insight on the impact of gravity change on thymic functions during spaceflight and living.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0141650PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4626100PMC
June 2016

Loss of Tifab, a del(5q) MDS gene, alters hematopoiesis through derepression of Toll-like receptor-TRAF6 signaling.

J Exp Med 2015 Oct 12;212(11):1967-85. Epub 2015 Oct 12.

Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH 45229 Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, OH 45267

TRAF-interacting protein with forkhead-associated domain B (TIFAB) is a haploinsufficient gene in del(5q) myelodysplastic syndrome (MDS). Deletion of Tifab results in progressive bone marrow (BM) and blood defects, including skewed hematopoietic stem/progenitor cell (HSPC) proportions and altered myeloid differentiation. A subset of mice transplanted with Tifab knockout (KO) HSPCs develop a BM failure with neutrophil dysplasia and cytopenia. In competitive transplants, Tifab KO HSPCs are out-competed by wild-type (WT) cells, suggesting a cell-intrinsic defect. Gene expression analysis of Tifab KO HSPCs identified dysregulation of immune-related signatures, and hypersensitivity to TLR4 stimulation. TIFAB forms a complex with TRAF6, a mediator of immune signaling, and reduces TRAF6 protein stability by a lysosome-dependent mechanism. In contrast, TIFAB loss increases TRAF6 protein and the dynamic range of TLR4 signaling, contributing to ineffective hematopoiesis. Moreover, combined deletion of TIFAB and miR-146a, two genes associated with del(5q) MDS/AML, results in a cooperative increase in TRAF6 expression and hematopoietic dysfunction. Re-expression of TIFAB in del(5q) MDS/AML cells results in attenuated TLR4 signaling and reduced viability. These findings underscore the importance of efficient regulation of innate immune/TRAF6 signaling within HSPCs by TIFAB, and its cooperation with miR-146a as it relates to the pathogenesis of hematopoietic malignancies, such as del(5q) MDS/AML.
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http://dx.doi.org/10.1084/jem.20141898DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4612089PMC
October 2015

Foxc2 in pharyngeal arch mesenchyme is important for aortic arch artery remodelling and ventricular septum formation.

Biomed Res 2015 ;36(4):235-45

Department of Biochemistry, Hamamatsu University School of Medicine.

The forkhead box C2 (Foxc2) protein is a member of the forkhead/winged helix transcription factor family and plays an essential role in cardiovascular development. Previous studies showed that Foxc2 null mouse embryos die during midgestation or just after birth with severe cardiovascular defects, including interruption, coarctation of the aortic arch and ventricular septal defects. These are also seen in human congenital heart disease. However, the tissue specific role of Foxc2 in aortic arch remodelling is not yet fully understood. Here we show that Foxc2 is expressed in a restricted pattern in several cell populations, including the mesenchyme and endothelium of pharyngeal arch arteries, which are important for cardiovascular development. In this study, we use a conditional knockout approach to examine the tissue specific role of Foxc2 in aortic arch remodelling. We demonstrate that mouse embryos lacking Foxc2 in Nkx2.5-expressing mesenchyme and endothelium of pharyngeal arch arteries display aortic arch interruption type B and ventricular septal defects. In contrast, conditional deletion of Foxc2 in Tie2-expressing endothelial cells does not result in aortic arch or ventricular septal defects, but does result in embryonic lethality due to peripheral oedema. Our data therefore provide for a detailed understanding of the role of mesenchymal Foxc2 in aortic arch remodelling and in the development of ventricular septum.
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http://dx.doi.org/10.2220/biomedres.36.235DOI Listing
May 2016

Calpain-Mediated Degradation of Drebrin by Excitotoxicity In vitro and In vivo.

PLoS One 2015 23;10(4):e0125119. Epub 2015 Apr 23.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

The level of drebrin, an evolutionarily conserved f-actin-binding protein that regulates synaptic structure and function, is reduced in the brains of patients with chronic neurodegenerative diseases such as Alzheimer's disease (AD) and Down's syndrome (DS). It was suggested that excitotoxic neuronal death caused by overactivation of NMDA-type glutamate receptors (NMDARs) occurs in AD and DS; however, the relationship between excitotoxicity and drebrin loss is unknown. Here, we show that drebrin is a novel target of calpain-mediated proteolysis under excitotoxic conditions induced by the overactivation of NMDARs. In cultured rodent neurons, degradation of drebrin was confirmed by the detection of proteolytic fragments, as well as a reduction in the amount of full-length drebrin. Notably, the NMDA-induced degradation of drebrin in mature neurons occurred concomitantly with a loss of f-actin. Furthermore, pharmacological inhibition of f-actin loss facilitated the drebrin degradation, suggesting a functional linkage between f-actin and drebrin degradation. Biochemical analyses using purified drebrin and calpain revealed that calpain degraded drebrin directly in vitro. Furthermore, cerebral ischemia also induced the degradation of drebrin in vivo. These findings suggest that calpain-mediated degradation of drebrin is a fundamental pathology of neurodegenerative diseases mediated by excitotoxicity, regardless of whether they are acute or chronic. Drebrin regulates the synaptic clustering of NMDARs; therefore, degradation of drebrin under excitotoxic conditions may modulate NMDAR-mediated signal transductions, including pro-survival signaling. Overall, the results presented here provide novel insights into the molecular basis of cellular responses to excitotoxicity in vitro and in vivo.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0125119PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4408054PMC
April 2016

The histone demethylase Fbxl11/Kdm2a plays an essential role in embryonic development by repressing cell-cycle regulators.

Mech Dev 2015 Feb 30;135:31-42. Epub 2014 Oct 30.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Methylation and de-methylation of histone lysine residues play pivotal roles in mammalian early development; these modifications influence chromatin architecture and regulate gene transcription. Fbxl11 (F-box and leucine-rich repeat 11)/Kdm2a is a histone demethylase that selectively removes mono- and di-methylation from histone H3K36. Previously, two other histone H3K36 demethylases (Jmjd5 or Fbxl10) were analyzed based on the phenotypes of the corresponding knockout (KO) mice; the results of those studies implicated H3K36 demethylases in cell proliferation, apoptosis, and senescence (Fukuda et al., 2011; Ishimura et al., 2012). To elucidate the physiological role of Fbxl11, we generated and examined Fbxl11 KO mice. Fbxl11 was expressed throughout the body during embryogenesis, and the Fbxl11 KO mice exhibited embryonic lethality at E10.5-12.5, accompanied with severe growth defects leading to reduced body size. Furthermore, knockout of Fbxl11 decreased cell proliferation and increased apoptosis. The lack of Fbxl11 resulted in downregulation of the Polycomb group protein (PcG) Ezh2, PcG mediated H2A ubiquitination and upregulation of the cyclin-dependent kinase inhibitor p21Cip1. Taken together, our findings suggest that Fbxl11 plays an essential role in embryonic development and homeostasis by regulating cell proliferation and survival.
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http://dx.doi.org/10.1016/j.mod.2014.10.001DOI Listing
February 2015

Runx2-I isoform contributes to fetal bone formation even in the absence of specific N-terminal amino acids.

PLoS One 2014 22;9(9):e108294. Epub 2014 Sep 22.

Division of Mucosal Immunology, Department of Microbiology and Immunology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; Department of Medical Genome Science, Graduate School of Frontier Science, The University of Tokyo, Chiba, Japan; Core Research for Evolutional Science and Technology (CREST), Japan Science and Technology Agency, Tokyo, Japan.

The Runt-related transcription factor 2 (Runx2) gene encodes the transcription factor Runx2, which is the master regulator of osteoblast development; insufficiency of this protein causes disorders of bone development such as cleidocranial dysplasia. Runx2 has two isoforms, Runx2-II and Runx2-I, and production of each isoform is controlled by a unique promoter: a distal promoter (P1) and a proximal promoter (P2), respectively. Although several studies have focused on differences and similarities between the two Runx2 isoforms, their individual roles in bone formation have not yet been determined conclusively, partly because a Runx2-I-targeted mouse model is not available. In this study, we established a novel Runx2-manipulated mouse model in which the first ATG of Runx2-I was replaced with TGA (a stop codon), and a neomycin-resistant gene (neo) cassette was inserted at the first intron of Runx2-I. Homozygous Runx2-Ineo/neo mice showed severely reduced expression of Runx2-I, whereas Runx2-II expression was largely retained. Runx2-Ineo/neo mice showed neonatal lethality, and in these mice, intramembranous ossification was more severely defective than endochondral ossification, presumably because of the greater involvement of Runx2-I, compared with that of Runx2-II in intramembranous ossification. Interestingly, the depletion of neo rescued the above-described phenotypes, indicating that the isoform-specific N-terminal region of Runx2-I is not functionally essential for bone development. Taken together, our results provide a novel clue leading to a better understanding of the roles of Runx2 isoforms in osteoblast development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0108294PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4171521PMC
December 2015

Neuromuscular disease. DOK7 gene therapy benefits mouse models of diseases characterized by defects in the neuromuscular junction.

Science 2014 Sep;345(6203):1505-8

Division of Genetics, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

The neuromuscular junction (NMJ) is the synapse between a motor neuron and skeletal muscle. Defects in NMJ transmission cause muscle weakness, termed myasthenia. The muscle protein Dok-7 is essential for activation of the receptor kinase MuSK, which governs NMJ formation, and DOK7 mutations underlie familial limb-girdle myasthenia (DOK7 myasthenia), a neuromuscular disease characterized by small NMJs. Here, we show in a mouse model of DOK7 myasthenia that therapeutic administration of an adeno-associated virus (AAV) vector encoding the human DOK7 gene resulted in an enlargement of NMJs and substantial increases in muscle strength and life span. When applied to model mice of another neuromuscular disorder, autosomal dominant Emery-Dreifuss muscular dystrophy, DOK7 gene therapy likewise resulted in enlargement of NMJs as well as positive effects on motor activity and life span. These results suggest that therapies aimed at enlarging the NMJ may be useful for a range of neuromuscular disorders.
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http://dx.doi.org/10.1126/science.1250744DOI Listing
September 2014

Development of FGF2-dependent pluripotent stem cells showing naive state characteristics from murine preimplantation inner cell mass.

Stem Cell Res 2014 Jul 26;13(1):75-87. Epub 2014 Apr 26.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

Two distinct types of embryonic pluripotent stem cells can be established from either the inner cell mass (ICM) of preimplantation blastocyst (leukemia inhibitory factor (LIF)-dependent embryonic stem cell, ESC, called naive state) or the epiblast of postimplantation fetuses (fibroblast growth factor 2 (FGF2)-dependent epiblast stem cells, EpiSC, called primed state). Here, we report that naive pluripotent stem cell was established from the ICM, but maintained its self-renewal by treatment with FGF2 and mouse embryonic fibroblasts (MEFs) when they were exposed FGF2 during establishment. This cell line is competent to contribute to chimeric animals, including germ cells, at high efficiency. The ERK1/2, SMAD2/3, and JAK/STAT3 pathways are essential to maintain self-renewal. Inhibition of ERK1/2 or SMAD2/3 initiates transition to a naive state ESC-like state, whereas inhibition of JAK/STAT3 promotes a primed EpiSC-like character. Our present results could provide novel insights into understanding the growth factor environment and ICM plasticity, and mechanisms which orchestrate the pluripotency of embryonic stem cells and the capacity for chimeric contributions.
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http://dx.doi.org/10.1016/j.scr.2014.04.012DOI Listing
July 2014

Mammalian-specific sequences in pou3f2 contribute to maternal behavior.

Genome Biol Evol 2014 Apr 7;6(5):1145-56. Epub 2014 Apr 7.

Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Japan

Various mutations have occurred during evolution among orthologs, genes in different species that diverged from a common ancestral gene by speciation. Here, we report the remarkable deterioration of a characteristic mammalian maternal behavior, pup retrieval, in nonmammalized mice, in which the transcription factor Pou3f2 was replaced with the Xenopus ortholog lacking all of the homopolymeric amino acid repeats of mammalian POU3F2. Most of the pups born to the nonmammalized mice died within days after birth, depending on the dam genotype alone. Quantitative immunohistochemical analysis revealed decreases in the rate-limiting enzymes of dopamine and serotonin synthesis in various brain structures. Similar results were obtained in knock-in mice in which all of the homopolymeric amino acid repeats of mammalian POU3F2 were removed. Pup retrieval behavior in mammals is thus strongly related to monoamine neurotransmitter levels via the acquisition of homopolymeric amino acid repeats during mammalian evolution.
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http://dx.doi.org/10.1093/gbe/evu072DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4040985PMC
April 2014

The Cd6 gene as a permissive locus for targeted transgenesis in the mouse.

Genesis 2014 May 17;52(5):440-50. Epub 2014 Apr 17.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

The introduction of a transgene into the genome through homologous recombination or sequence-specific enzymatic modification is a key technique for producing transgenic mice. The Rosa26 gene has been widely used to produce transgenic mice because the gene is transcriptionally active in various cell types and, at many developmental stages, is permissive for constitutive expression of integrated transgenes, and is dispensable for normal development. However, permissive loci other than Rosa26 are needed to generate mice that harbor multiple transgenes for complex studies. Here, we identified the Cd6 locus on mouse chromosome 19 as a transgene integration site in a transgenic mouse strain showing widespread reporter expression. Using this locus, we generated a knock-in mouse line that harbors a CAG promoter-driven reporter transgene, and found that the homozygous transgenic mice are viable and fertile, although transgene insertion disrupted Cd6 gene expression. The transgene on the Cd6 locus expressed reporter genes extensively throughout embryos, neonates, and adults. Combined with the Cre/loxP binary system, blood and lymphatic endothelial cell-specific reporter expression from the transgenic locus was achieved. These results suggest that Cd6 is valuable as an alternative site for targeted transgenesis.
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http://dx.doi.org/10.1002/dvg.22779DOI Listing
May 2014

Lnk prevents inflammatory CD8⁺ T-cell proliferation and contributes to intestinal homeostasis.

Eur J Immunol 2014 Jun 2;44(6):1622-32. Epub 2014 May 2.

Department of Immune Regulation, Research Institute, National Center for Global Health and Medicine, Ichikawa, Chiba, Japan.

The intracellular adaptor Lnk (also known as SH2B3) regulates cytokine signals that control lymphohematopoiesis, and Lnk(-/-) mice have expanded B-cell, megakaryocyte, and hematopoietic stem-cell populations. Moreover, mutations in the LNK gene are found in patients with myeloproliferative disease, whereas LNK polymorphisms have recently been associated with inflammatory and autoimmune diseases, including celiac disease. Here, we describe a previously unrecognized function of Lnk in the control of inflammatory CD8(+) T-cell proliferation and in intestinal homeostasis. Mature T cells from newly generated Lnk-Venus reporter mice had low but substantial expression of Lnk, whereas Lnk expression was downregulated during homeostatic T-cell proliferation under lymphopenic conditions. The numbers of CD44(hi) IFN-γ(+) CD8(+) effector or memory T cells were found to be increased in Lnk(-/-) mice, which also exhibited shortening of villi in the small intestine. Lnk(-/-) CD8(+) T cells survived longer in response to stimulation with IL-15 and proliferated even in nonlymphopenic hosts. Transfer of Lnk(-/-) CD8(+) T cells together with WT CD4(+) T cells into Rag2-deficient mice recapitulated a sign of villous abnormality. Our results reveal a link between Lnk and immune cell-mediated intestinal tissue destruction.
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http://dx.doi.org/10.1002/eji.201343883DOI Listing
June 2014

FGF2-induced Ras-MAPK signalling maintains lymphatic endothelial cell identity by upregulating endothelial-cell-specific gene expression and suppressing TGFβ signalling through Smad2.

J Cell Sci 2014 Feb 19;127(Pt 4):845-57. Epub 2013 Dec 19.

Laboratory of Developmental Genetics, Center for Experimental Medicine and Systems Biology, The Institute of Medical Science, the University of Tokyo, Minato-ku, Tokyo 108-8639, Japan.

The lymphatic endothelial cell (LEC) fate decision program during development has been described. However, the mechanism underlying the maintenance of differentiated LEC identity remains largely unknown. Here, we show that fibroblast growth factor 2 (FGF2) plays a fundamental role in maintaining a differentiated LEC trait. In addition to demonstrating the appearance of LECs expressing α-smooth muscle actin in mouse lymphedematous skin in vivo, we found that mouse immortalised LECs lose their characteristics and undergo endothelial-to-mesenchymal transition (EndMT) when cultured in FGF2-depleted medium. FGF2 depletion acted synergistically with transforming growth factor (TGF) β to induce EndMT. We also found that H-Ras-overexpressing LECs were resistant to EndMT. Activation of H-Ras not only upregulated FGF2-induced activation of the Erk mitogen activated protein kinases (MAPK3 and MAPK1), but also suppressed TGFβ-induced activation of Smad2 by modulating Smad2 phosphorylation by MAPKs. These results suggest that FGF2 regulates LEC-specific gene expression and suppresses TGFβ signalling in LECs through Smad2 in a Ras-MAPK-dependent manner. Taken together, our findings provide a new insight into the FGF2-Ras-MAPK-dependent mechanism that maintains and modulates the LEC trait.
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http://dx.doi.org/10.1242/jcs.137836DOI Listing
February 2014