Recent development of transgenic techniques has made it possible to directly ana-lyze the functions of a particular gene in a living animal. These techniques havealso made it possible to produce various animal disease models as well as toolsto analyze them. Immune disorders and infectious diseases are our major con-cerns, and we are attempting to produce transgenic mouse models for these dis-eases.

1. Studies on rheumatoid arthritis models:human T cell leukemia virus type I (HTLV-I) transgenic (Tg) mouse model and IL-1receptor antagonist (IL-1Ra)-deficient (KO)mouse model

Shinobu Saijo, Noriyuki Fujikado, Soo-hyunChung, Kazusuke Shimamori, Keiko Yamabe,and Yoichiro Iwakura

Rheumatoid arthritis (RA) is one of the mostserious medical problems worldwide with ap-proximately 1％ of the people in the world af-fected. The disease is autoimmune in nature andcharacterized by chronic inflammation of thesynovial tissues in multiple joints that leads tojoint destruction. High levels of autoantibodiesin the serum and augmentation of proinflamma-tory cytokine expression in the joints are charac-teristics of the disease, although the pathogene-sis has not been elucidated completely. We havebeen studying the pathogenesis of the diseaseusing two arthritis models that we originally de-veloped. One is the HTLV-I Tg mouse model(Iwakura et al., Science, 1991) and the other is IL-1Ra KO (Horai et al., J. Exp. Med., 2000). Bothof these models develop autoimmunity and

chronic inflammatory arthropathy closely resem-bling RA in humans.

To identify genes involved in the pathogene-sis of arthritis, we analyzed the gene expressionprofiles of these animal models by using high-density oligonucleotide arrays. We found 1,467genes that were differentially expressed fromthe normal control mice by greater than three-fold in one of these animal models. The gene ex-pression profiles of the two models correlatedwell. We extracted 554 genes whose expressionsignificantly changed in both models, assumingthat pathogenically important genes at the effec-tor phase would change in both models. Then,each of these commonly changed genes wasmapped into the whole genome in a scale of the1-megabase pairs. We found that the transcrip-tome map of these genes did not distributeevenly on the chromosome but formed clusters.These identified gene clusters include the majorhistocompatibility complex class I and class IIgenes, complement genes, and chemokine genes,which are well known to be involved in thepathogenesis of RA at the effector phase. Theactivation of these gene clusters suggests thatantigen presentation and lymphocyte chemo-taxis are important for the development of ar-

Center for Experimental Medicine

Laboratory of Cell Biology細胞機能研究分野

Professor Yoichiro Iwakrua, D. Sc.Research Associate Dai Chida, Ph. D.Research Associate Shigeru Kakuta, Ph. D., D.V.M.

教 授 理学博士 岩 倉 洋一郎助 手 理学博士 千 田 大助 手 獣医学博士 角 田 茂

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thritis. Moreover, by searching for such clusters,we could detect genes with marginal expressionchanges. These gene clusters include schlafenand membrane-spanning four-domains subfam-ily A genes whose function in arthritis has notyet been determined. Thus, by combining twoetiologically different RA models, we succeededin efficiently extracting genes functioning in thedevelopment of arthritis at the effector phase.Furthermore, we demonstrated that identifica-tion of gene clusters by transcriptome mappingis a useful way to find potentially pathogenicgenes among genes whose expression change isonly marginal.

To examine the roles for these genes in thedevelopment of arthritis, we produced gene tar-geted mice of these genes and found that one ofthem was resistant to collagen induced arthritis,and the other one was more sensitive than thewild-type mice. Thus, generation of gene tar-geted mice of the genes identified by microarrayanalysis of these arthritic models is useful toidentify genes important for the development ofarthritis. We are now analyzing the function ofthe genes that are activated in the arthritic jointsby generating gene targeted mice of these genes.

2. Analysis of the roles of IL-17 family genesin the immune system

Harumichi Ishigame, Yutaka Komiyama, YukoTanahashi, Susumu Nakae, Taizo Matsuki,Aya Nambu, Shigeru Kakuta, Katsuko Sudo,and Yoichiro Iwakura

IL-17 is a proinflammatory cytokine that acti-vates T cells and other immune cells to producea variety of cytokines, chemokines, and cell ad-hesion molecules. This cytokine is augmented inthe sera and/or tissues of patients with contactdermatitis, asthma, and rheumatoid arthritis.We previously demonstrated that IL-17 is in-volved in the development of autoimmune ar-thritis and contact, delayed, and airway hyper-sensitivity in mice. Recently, it was shown thatIL-17 is produced by a newly discovered CD4＋

T cell subset, Th17. Now, many diseases thatwere though to be induced by Th1 or Th2 areproven to be induced by Th17. Thus, we areanalyzing the roles of IL-17 and its family mole-cules in inflammatory diseases and host defensemechanism against infection.

As the expression of IL-17 is augmented inmultiple sclerosis (MS), we examined the in-volvement of this cytokine in these diseases us-ing IL-17－/－ murine disease models. We foundthat the development of experimental autoim-mune encephalomyelitis (EAE), the rodentmodel of MS, was significantly suppressed in IL

-17－/－ mice; these animals exhibited a delayedonset, a reduced maximum severity scores, ame-liorated histological changes, and early recovery.T cell sensitization against myelin oligodendro-cyte glycoprotein was reduced in IL-17－/－ miceupon sensitization. The major producer of IL-17upon treatement with MOG was CD4＋ T cellsrather than CD8＋ T cells, and adoptive transferof IL-17－/－ CD4＋ T cells inefficiently inducedEAE in recipient mice. Notably, IL-17-producingT cells were increased in IFN-γ－/－ cells, whileIFN-γ-producing cells were increased in IL-17－/－cells, suggesting that IL-17 and IFN-γ mutuallyregulate IFN-γ and IL-17 production. These ob-servations indicate that IL-17 rather than IFN-γplays a crucial role in the development of EAE.

3. Dectin-1 is required for host defenseagainst Pneumocystis carinii, but not Can-dida albicans

Shinobu Saijo, Noriyuki Fujikado, TakahisaFuruta1, Soo-hyun Chung, Hayato Kotaki, Kei-suke Seki, Katsuko Sudo, Shizuo Akira2, Yoshi-yuki Adachi3, Naohito Ohno3, Takeshi Kinjo4,Kiwamu Nakamura4, Kazuyoshi Kawakami5,and Yoichiro Iwakura: 1Department of Micro-biology and Immunology, Institute for MedicalScience, University of Tokyo; 2Department ofHost Defense, Research Institute for MicrobialDisease, Osaka University; 3Laboratory for Im-munopharmacology of Microbial Products,School of Pharmacy, Tokyo University of Phar-macy and Life Science; 4Department of Medi-cine and Therapeutics, Control and Preventionof Infectious Diseases, Faculty of Medicine,University of the Ryukyu; 5Microbiology andImmunology, Department of Medical Technol-ogy, School of Health Science, Faculty of, To-hoku University

Dectin-1 is a C-type lectin involved in the rec-ognition of β-glucans found in cell walls offungi. We generated Dectin1-deficient mice toexamine the importance of Dectin-1 in defenseagainst pathogenic fungi. In vitro, β-glucan-induced cytokine production from wild-typedendritic cells (DCs) and macrophages wasabolished in Dectin1－/－ cells. In vivo, comparedto wild-type mice, Dectin1－/－ mice were moresusceptible to pneumocystis infection eventhough cytokine production was normal. How-ever, pneumocystis-infected Dectin1－/－ macro-phages did demonstrate defective production ofreactive oxygen. In contrast those results, wild-type and Dectin1－/－ mice were equally suscepti-ble to candida infection. Thus, Dectin-1 is re-quired for immune responses to some fungal in-fection, as protective immunity to pneumocystis,

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but not candida, requires Dectin-1 for produc-tion of anti-fungal reactive oxygen.

4. The role of cytokines in the neuro-immuno-endocrine system

Dai Chida, Toshimasa Osaka6, Okito Hashi-moto, Chisato Wakabayashi, and YoichiroIwakura: 6National Institute of Health and Nu-trition

Proinflammatory cytokines including IL-1 andIL-6 exert pleiotropic effects on the neuro-immuno-endocrine system. Previously , weshowed that IL-1 receptor antagonist deficient(IL-1Ra－/－) mice show a lean phenotype due toan abnormal lipid metabolism. On the contrary,it was reported that IL-6－/－ mice exhibit obesityafter 6 months of age. This study sought to as-sess the roles of IL-1 and IL-6 in body weighthomeostasis. We generated mice deficient in IL-6 and IL-1Ra (IL-6－/－IL-1Ra－/－), and IL-6, IL-1α,and IL-1β (IL-6－/－IL-1－/－). IL-6－/－IL-1Ra－/－ miceexhibited a lean phenotype, similarly to IL-1Ra－/－mice. On the other hand, IL-6－/－IL-1－/－

mice became obese as early as 10 weeks of age,while IL-1－/－ mice and IL-6－/－ mice were nor-mal at this age. The daily food intake was sig-nificantly higher in IL-6－/－IL-1－/－ mice than inIL-6－/－IL-1＋/－ mice, while energy expenditurewas comparable in these two strains. Acute ano-rexia induced by peripheral administration of IL-1 was significantly suppressed in IL-6－/－IL-1－/－

mice, but not in IL-1－/－ mice or IL-6－/－ mice, ascompared to WT mice. These results indicatethat IL-1 and IL-6 are both involved in the regu-lation of body fat in a redundant manner inyoung mice. We are now analyzing the roles ofIL-1 in the central nervous system, because IL-1is induced in the brain upon exposure to stressand affects various neuronal functions such asappetite, sleep and emotion. IL-1－/－ and IL1Ra－/－ mice should be useful to analyze the func-tions of these cytokines in the neuronal system.

5. Studies on 2’, 5’-oligoadenylate synthetaseand inflammation related genes

Shigeru Kakuta, Reiko Ichikawa, Satoe Azechi,Mari Shibukawa, Hayato Kotaki, and YoichiroIwakura

2’, 5’-oligoadenylate synthetase (OAS) is oneof IFN-inducible proteins and involved in theantiviral mechanism through activation ofRNaseL. In humans, there are four classes ofOAS genes, short (OAS1), middle (OAS2), andlong (OAS3) form genes and one OAS-like pro-tein (OASL) gene. On the other hand, in mice,

we previously showed that the OAS1 gene ismultiplicated into 8 genes and the OASL gene isduplicated. The short form OAS has a set of theessential motifs for the OAS enzyme activity,OAS2 has two and OAS3 has three catalyticunits. OASL has a single OAS unit and two con-secutive ubiquitin-like sequences in the carboxyl-terminal, but lacks OAS activity. The physi-ological roles and functional difference amongthese genes, however, are not elucidated com-pletely. In this study, to elucidate the roles ofOAS family genes, we generated each of Oas1a- ,Oas1c-, Oas2-, Oas1a/Oas2-, and Oas1-3 genecluster (OASC )-deficient mice with targetedgene disruptions.

Oas1a/Oas2 deficient (flox) mice have two loxPsites inserted into the flanking regions of theOas gene cluster. A large size deletion (～170kb)between loxP sites was created by a Cre-mediated recombination. OASC -deficient micewere generated by injecting a Cre expressionplasmid, pCAG-Cre, into 1-cell embryos derivedfrom Oas1a/Oas2-deficient (flox) mice. OASCmice were born in a Mendelian ratio, fertile, andappeared healthy under SPF conditions. We arenow analyzing the effects of these gene deficien-cies on the host defense against viral infectionand various inflammatory responses includingConcanavalin A-induced hepatitis. We are alsoanalyzing the roles of genes that are activated inthe joints of rheumatoid arthritis models in al-lergic responses and tumor development.

6. Generation of AIDS disease models andanalysis of the pathogenesis using animalmodels

Naomi Tsurutani , Jiro Yasuda , NaokiYamamoto7, Byung-Il Choi, Motohiko Kadoki,Kuniaki Yabe, Takuya Tada, and YoichiroIwakura: 7Department of Molecular Virology,School of Medicine, Tokyo Medical and DentalUniversity

Studies of AIDS pathogenesis and develop-ment of therapeutic drugs for AIDS have beenhampered by the lack of appropriate small ani-mal models for AIDS, because human immu-nodeficiency virus type-1 (HIV-1) cannot infectand replicate in mice because the structure ofsome host factors necessary for HIV-1 replica-tion is different between mice and humans. Asprimate models are difficult to handle, expen-sive and require expensive facilities, geneticallynot well characterized, often infected withpathogens and sometimes dangerous, and ethi-cally less permissible, we are trying to generatemouse models for AIDS. We are taking two ap-proaches to generate the AIDS models, one is

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HIV-1 carrier models which carry the HIV-1genome as a transgene (Iwakura et al., AIDS ,1992) and the other is HIV-1 susceptible modelsin which all the host factors involved in the spe-cies barrier are humanized.

Mouse cells do not support HIV-1 replicationbecause of host range barriers at steps includingvirus entry, transcription, RNA splicing ,polyprotein processing, assembly, and release.The exact mechanisms for the suppression, how-ever, are not completely understood. To eluci-date further the barriers against HIV-1 replica-tion in mouse cells, we analyzed the replicationof the virus in lymphocytes from human CD4/CXCR4 transgenic mice. Although primarysplenocytes and thymocytes allowed the entryand reverse transcription of HIV-1, the integra-tion efficiency of the viral DNA was greatly re-duced in these cells relative to human periph-eral blood mononuclear cells (PBMCs), suggest-ing an additional block(s) before or at the pointof host chromosome integration of the viralDNA. Pre-integration processes were furtheranalyzed using HIV-1 pseudotyped viruses. Thereverse transcription step of HIV-1 pseudotypedwith the envelope of murine leukemia virus(MuLV) or vesicular stomatitis virus glycopro-tein (VSV-G) was efficiently supported in bothhuman and mouse cells, but nuclear import ofthe pre-integration complex (PIC) of HIV-1 wasblocked in mouse cells. We found that greenfluorescent protein (GFP)-labeled HIV-1 inte-grase, which is known to be important in thenuclear localization of the PIC, could not be im-ported into the nucleus of mouse cells, in con-trast to human cells. On the other hand, GFP-Vpr localized exclusively to the nuclei of bothmouse and human cells. These observations sug-gest that, due to the dysfunction of integrase,the nuclear localization of PIC is suppressed inmouse cells. We are now analyzing the host fac-tors which interact with integrase and trying togenerate transgenic mice carrying the humangene for this host barrier factor. In another at-tempt, we are further analyzing the host barriersteps and identifying the responsible genes.

7. Studies on the molecular mechanismsfunctioning in the early development ofmouse embryos

Kenjiro Adachi, Seiji Takashima, HiroakiOkae, and Yoichiro Iwakura

Blastocyst formation during mammalian pre-implantation development is a unique develop-mental process that involves lineage segregationbetween the inner cell mass and the trophecto-derm. To elucidate the molecular mechanismsunderlying blastocyst formation, we have func-tionally screened a subset of preimplantationembryo-associated transcripts using siRNA andidentified Bysl (bystin-like) as an essential genefor this process. The development of embryosinjected with Bysl siRNA was arrested just priorto blastocyst formation, resulting in a defect introphectoderm differentiation. Silencing of Byslusing an episomal shRNA expression vector in-hibited proliferation of embryonic stem cells. Ex-ogenously expressed Bysl tagged with a fluores-cent protein was concentrated in the nucleoluswith a diffuse nucleoplasmic distribution. Fur-thermore, loss of Bysl function using RNAi ordominant negative mutants caused defects in 40S ribosomal subunit biogenesis. These findingsprovide evidence for a crucial role of Bysl as anintegral factor for ribosome biogenesis, and sug-gest a critical dependence of blastocyst forma-tion on active translation machinery.

As our previous studies suggest that cell-cellinteraction during 8 cell compaction stage is im-portant for the cell lineage determination duringblastocyst formation, we are further trying toidentify these lineage determination responsiblegenes which are involved in cell-cell interactionusing in silico screening and RNAi techniques.We have isolated several candidate genes andare now analyzing the functions of these genes.In another approach, we are trying to identifythese lineage determination responsible genesusing a secretary trap method. We screened ap-proximately 1000 trapped ES clones. From theseclones, we obtained 34 clones that are specific toES cells and encode membrane proteins. Amongthese clones, 12 genes were already mutated inmice, of which 10 showed recessive lethal phe-notype, indicating that this screening method isextremely effective to enrich developmentallyimportant genes. We are now trying to producethe mutant mice from the residual clones.

Publications

Matsuki, T., Nakae, S., Sudo, K., Horai, R., andIwakura, Y. Abnormal T cell activation causedby the imbalance of the IL-1/IL-1 receptor an-tagonist system is responsible for the develop-ment of experimental autoimmune encephalo-

myelitis. Int. Immunol ., 18, 399-407 (2006).Ishigame, H., Nakajima, A., Saijo, S., Komiyama,

Y., Mastuki, T., Nakae, S., Horai, R., Kakuta,S., and Iwakura, Y. The role of TNFα and IL-17 in the development of excess IL-1 signaling

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-induced inflammatory diseases in IL-1 recep-tor antagonist-deficient mice. Ernst ScheringRes. Found. Workshop , 56, 129-153 (2006).

Shimizu, M., Shimamura, M., Owaki, T., Asak-awa, M., Fujita, K., Kudo, M., Iwakura, Y.,Takeda, Y., Mizuguchi, J., and Yoshimoto, T.Antiangiogenic and antitumor activities of IL-27. J. Immunol ., 176, 7317-7324 (2006).

Honore, P., Wade, C.L., Zhong, C., Harris, R.R.,Wu, C., Ghayur, T., Iwakura, Y., Decker, M.W., Faltynek, C., Sullivan, J., Jarvis, M.F.Interleukin-αβ gene-deficient mice show re-duced nociceptive sensitivity in inflammatoryand neuropathic pain but not post-operativepain. Behav. Brain Res., 167, 355-364 (2006).

Kotani, M., Hirata, K., Ogawa, S., Habiro, K.,Araki, M., Ishi, T., Ishida, Y., Tanuma, S., Tan-abe, K., Toma, Horai, R., Iwakura, Y., andAbe, R. Presence of CD28-dependent and in-dependent pathways in autoimmune arthritisdeveloped in interleukin-1 receptor antagonist-deficient mice. Arth. Rheum ., 54, 473-481(2006).

Deng, X., Yu, Z., Funayama, H., Shoji, N.,Sasano, T., Iwakura, Y., Sugawara, S., andEndo, Y. Mutual augmentation of the induc-tion of the histamine-forming enzyme, his-tidine decarboxylase, between alendronateand immuno-stimulants (IL-1, TNF, and LPS),and its prevention by clodronate. Toxicol.Appl. Pharmacol ., 213, 64-73 (2006).

Hadano, S., Benn, S., Kakuta, S., Otomo, A.,Sudo, K., Kunita, R., Suzuki-Utsunomiya, K.,Mizumura, H., Shefner, J.M., Cox, G.A.,Iwakura, Y., Brown, R.H. Jr., and Ikeda, J.Loss of ALS2 in mice produces mild, age-dependent loss of cerebellar Purkinje cells andsubtle pathology of large spinal motor neu-rons without an obvious motor phenotype.Human Molecular Genetics , 15, 233-250 (2006).

Zhu, Y., Saito, K., Murakami, Y., Asano, M.,Iwakura, Y., and Seishima,M. Early increase inmRNA levels of pro-inflammatory cytokinesand their interactions in the mouse hippocam-pus after transient global ischemia. Neurosci-ence Letter, 393, 122-126 (2006).

Vonk, A.G., Netea, M.G., van Krieken, J.H.,Iwakura, Y., van der Meer, J.W.M., and Kull-berg, J.B. Endogenous interleukin-1α andinterleukin-1β are crucial for host defenseagainst disseminated candidiasis. J. Infect.Dis ., 193, 1419-1426 (2006).

Chida, D., Osaka, T., Hashimoto, O., andIwakura, Y. Combined IL-6 and IL-1 defi-ciency causes obesity in young mice. Diabetes ,55, 971-977 (2006).

Nambu, A., Nakae, S., and Iwakura, Y. IL-1β,but not IL-1α, is required for antigen-specificT cell activation and the induction of local in-

flammation in the delayed-type hypersensitiv-ity responses. Int. Immunol ., 18, 701-712(2006).

Sasaki, S., Tagawa, Y., Iwakura, Y., and Nakane,A. The role of gamma interferon in acquiredhost resistance against Staphylococcus aureusinfection in mice. FEMS Immunol. Med. Micro-biol ., 46, 367-374 (2006).

Tateno, K., Minamino, T., Toko, H., Akazawa,H., Shimizu, N., Takeda, S., Kunieda, T., Mi-yauchi, H., Oyama, T., Matsuura, K., Nishi, J.I., Kobayashi, Y., Nagai, T., Kuwabara, Y.,Iwakura, Y., Nomura, F., Saito, Y., andKomuro, I. Critical roles of muscle-secretedangiogenic factors in therapeutic neovasculari-zation. Circ. Res ., 98, 1194-1202 (2006).

O’Sullivan, B.J., Thomas, H.E.,Pai, S., San-tamaria, P., Iwakura, Y., Steptoe, R.J., Kay, T.W.H., and Thomas, R. IL-1β breaks tolerancethrough expansion of CD25＋ effector T cells. J.Immunol ., 176, 7278-7287 (2006).

Iwakura, Y., and Ishigame, H. The IL-23/IL-17axis in inflammation. J. Clin. Invest ., 116, 1218-1222 (2006).

Ohtaki, H., Nakamachi, T., Dohi, K., Aizawa, Y.,Takaki, A., Hodoyama, K., Yofu, S., Hashi-moto, H., Shintani, N., Baba,A., Kopf, M.,Iwakura, Y., Matsuda, K., Arimura, A., andShioda , S . Pituitary adenylate cyclase-activating polypeptide (PACAP) decreasesischemic neuronal cell death in associationwith interleukin-6. Proc. Natl. Acad. Sci. USA,103, 7488-7493 (2006).

Komiyama, Y., Nakae, S., Matsuki, T., Nambu,A., Ishigame, H., Kakuta, S., Sudo, K., andIwakura, Y. IL-17 plays an important role inthe development of experimental autoimmuneencephalomyelitis. J. Immunol ., 177, 566-573(2006).

Uetani, N., Chagnon, M.J., Kennedy, T.E.,Iwakura, Y., and Tremblay, M.L. Mammalianmotoneuron axon targeting requires receptorprotein tyrosine phosphatases sigma anddelta. J. Neurosci ., 26, 5872-5880 (2006).

Nakazawa, T., Komai, S., Watabe, A.M., Ki-yama, Y., Fukaya, M., Arima-Yoshida, F.,Horai, R., Sudo, K., Ebine, K., Delawary, M.,Goto, J., Umemori, H., Tezuka, T., Iwakura,Y., Watanabe, M., Yamamoto, T., and Manabe,T. NR2B tyrosine phosphorylation modulatesfear learning as well as amygdaloid synapticplasticity. EMBO J ., 25, 2867-2877 (2006).

Murakami, M., Iwai, S., Hiratsuka, S., Yamauchi,M., Nakamura, K., Iwakura, Y., and Shibuya,M. Signaling of vascular endothelial growthfactor receptor-1 tyrosine kinase promotesrheumatoid arthritis through activation ofmonocyte/macrophage. Blood , 108, 1849-1856(2006).

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Okada, K., Inoue, A., Okada, M., Murata, Y.,Kakuta, S., Jigami, T., Shiraishi, H., Eguchi, K.,Motomura, M., Akiyama, T., Iwakura, Y.,Higuchi, O., and Yamanashi, Y. The muscleprotein Dok-7 is essential for neuromuscularsynaptogenesis. Science, 312, 1802-1805 (2006).

Fujikado, N., Saijo, S., and Iwakura, Y. Identifi-cation of arthritis-related gene clusters by mi-croarray analysis of two independent mousemodels for rheumatoid arthritis. Arthritis Res.Ther ., 8, R100, 1-13 (2006).

Furuta, T., Kikuchi, T., Iwakura, Y., and Watan-abe, N. Protective roles of mast cells and mastcell-derived tumor necrosis factor in murinemalaria. J. Immunol ., 177, 3294-302 (2006).

Voronov, E., Dayan, M., Zinger, H., Gayvoron-sky, L., Lin, J.P., Iwakura, Y., Apte, R.N., andMozes, E. IL-1beta-deficient mice are resistantto induction of experimental SLE. Eur. Cy-tokine Netw., 17, 109-16 (2006).

Furuichi, K., Wada, T., Iwata, Y., Kokubo, S.,Hara, A., Yamahana, J., Sugaya, T., Iwakura,Y., Matsushima, K., Asano, M., Yokoyama, H.,and Kaneko, S. Interleukin-1-dependent se-quential chemokine expression and inflamma-tory cell infiltration in ischemia-reperfusioninjury. Crit. Care Med ., 34, 2447-2455 (2006).

Konishi, Y., Ikeda, K., Iwakura, Y., andKawakami, K. Six1 and Six4 promote survivalof sensory neurons during early trigeminalgangliogenesis. Brain Res., 1116, 93-102 (2006).

Maedler, K., Schumann, D.M., Sauter, N.,Ellingsgaard, H., Bosco, D., Baertshiger, R.,Iwakura, Y., Oberholzer, J., Wollheim, C.B.,Gauthier, B.R., and Donath, M.Y. Low concen-tration of interleukin-1β induces FLICE-inhibitory protein-mediated β-cell prolifera-tion in human pancreatic islets. Diabetes , 55,2713-2722 (2006).

Sato, K., Suematsu, A., Okamoto, K., Yama-guchi, A., Morishita, Y., Kadono, Y., Tanaka,S., Kodama, T., Shizuo, A., Iwakura, Y., Cua,D.J., and Takayanagi, H. TH17 functions as anosteoclastogenic helper T cell subset that linksT cell activation and bone destruction. J. Exp.Med ., 203, 2673-2682 (2006).

Ito, R., Shin-Ya, M., Kishida, T., Urano, A.,Takada, R., Sakagami, J., Imanishi, J., Kita, M.,Ueda, Y., Iwakura, Y., Kataoka, K., Okanoue,T., and Mazda, O. Interferon-gamma is causa-tively involved in experimental inflammatorybowel disease in mice. Clin. Exp. Immunol .,146, 330-338 (2006).

Kamari, Y., Werman-Venkert, R., Shaish, A.,Werman, A., Harari, A., Gonen, A., Voronov,E., Grosskopf, I., Sharabi, Y., Grossman, E.,Iwakura, Y., Dinarello, C.A., Apte, R.N., andHarats, D. Differential role and tissue specific-ity of interleukin-1alpha gene expression inatherogenesis and lipid metabolism. Athero-sclerosis , in press.

Sugihara, T., Sekine, C., Nakae, T., Kohyama, K.,Harigai, M., Iwakura, Y., Matsumoto, Y., Miy-asaka, N., and Kohsaka, H. A new murinemodel defines critical mediators in the pathol-ogy and treatment of polymyositis. ArthritisRheum ., in press.

Nakae, S., Iwakura, Y., Suto, H., and Galli, S.J.Phenotypic differences between Th1 and Th17cells and negative regulation of Th1 cell-differentiation by IL-17. J. Leu. Biol ., in press.

角田茂．ノックアウトマウスの岩倉研式集団一括管理術．JSI Newsletter１４（１）：２２，２００６．

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藤門範行，岩倉洋一郎．関節リウマチの疾患モデル．治療学．ライフサイエンス出版．４０（７）：２５―２８，２００６．

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Gene targeting technology has revealed many aspects of gene functions in vivo.Knock out mice offer the opportunities of not only analyzing the complex genefunctions in vivo, but also presenting various human disease models, where newtherapeutic approaches can be explored. To allow more detailed dissection ofgene function, we introduce a point mutation or disrupt genes in certain lineages(or stages) using Cre-loxP system, a method of conditional gene targeting. In theprocess of analyzing knock out mice, we have isolated spontaneous mutant micewhich develop chylous ascites and edematous limbs. In order to understand themechanism of lymphatic development and functions in more detail, we are alsogenerating various knock-out/knock-in mouse lines including a conditional knockout mouse. ES cells, which are used for gene targeting, are the only stem cellsbeing cultured in vitro. To elucidate the molecular mechanism regulating self-renewal of pluripotent ES cells, we have tried to identify a factor(s) cooperatingwith Oct-3/4, the critical transcription factor for maintaining undifferentiated state ofES cells.

1. Morphological Change Caused by Loss ofthe Taxon-Specific Polyalanine Tract inHoxd-13.

Keiichi Anan1 , Nobuaki Yoshida , YukiKataoka, Mitsuharu Sato, Hirotake Ichise,Makoto Nasu1 and Shintaro Ueda1: 1Labora-tory for Molecular Anthropology and Molecu-lar Evolution, University of Tokyo.

Sequence comparison of Hoxd-13 among ver-tebrates revealed the presence of taxon-specificpolyalanine tracts in amniotes. To investigatetheir function at the organismal level, we re-placed the wild-type Hoxd-13 gene with one

lacking the 15-residue polyalanine tract by usinghomologous recombination. Sesamoid bone for-mation in knock-in mice was different from thatin the wild type; this was observed not only inthe homozygotes but also in heterozygotes. Thepresent study provides the first direct evidencethat taxon-specific homopolymeric amino acidrepeats are involved in phenotypic diversifica-tion at the organismal level.

2. Azoospermia in mice with targeted disrup-tion of the Brek/Lmtk2 (brain-enrichedkinase/lemur tyrosine kinase 2) gene.

Seiji Kawa2, Chizuru Ito3, Yoshiro Toyama3,

Center for Experimental Medicine

Laboratory of Gene Expression & Regula-tion遺伝子機能研究分野

Professor Nobuaki Yoshida, M.D., D.M.Sc.Research Associate Mitsuharu Sato, Ph.D.Research Associate Hirotake Ichise, D.V.M., Ph.D.Research Associate Taeko Ichise, Ph.D.

教 授 医学博士 吉 田 進 昭助 手 医学博士 佐 藤 充 治助 手 獣医学博士 市 瀬 広 武助 手 医学博士 市 瀬 多恵子

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Mamiko Maekawa3, Tohru Tezuka2, TakahisaNakamura2, Takanobu Nakazawa2, KazumasaYokoyama2, Nobuaki Yoshida, Kiyotaka Toshi-mori3, and Tadashi Yamamoto2: 2Division ofOncology, Institute of Medical Science, Univer-sity of Tokyo, 3Department of Anatomy andDevelopmental Biology, Graduate School ofMedicine, Chiba University.

Brek/Lmtk2 (brain-enriched kinase/lemur ty-rosine kinase 2) is a member of the Aatyk fam-ily of kinases that comprises Aatyk1, Brek/Lmtk2/Aatyk2, and Aatyk3. Although several poten-tial roles have been proposed for Brek and otherAatyk family members, the physiological func-tions of these kinases remain unclear. Here, wereport that Brek－/－ male mice are infertile, withazoospermia. Detailed histological analysis re-vealed that Brek－/－ germ cells differentiated nor-mally until the round-spermatid stage, butfailed to undergo the normal change in mor-phology to become elongated spermatids. Tes-ticular somatic cells appeared normal in thesemice. Expression of Brek in testis was restrictedto the germ cells, suggesting that the matura-tions of germ cells in Brek－/－ mice are affectedin a cell-autonomous manner. On the basis ofthese findings, we concluded that Brek is essen-tial for a late stage of spermatogenesis. Furtherclarification of the mechanism by which Brekregulates spermatogenesis may help identifynew targets for reproductive contraceptives andtreatments against infertility.

3. Efficient sequential gene regulation viaFLP- and Cre-recombinase using adenovi-rus vector in mammalian cells includingmouse ES cells.

Saki Kondo4, Yuzuka Takahashi4, Seiji Shio-zawa, Hirotake Ichise, Nobuaki Yoshida, YumiKanegae4, and Izumi Saito4: 4Laboratory ofMolecular Genetics, Institute of Medical Sci-ence, University of Tokyo.

Site-specific recombinase is widely applied forthe regulation of gene expression because itsregulatory action is strict and efficient. How-ever, each system can mediate regulation ofonly one gene at a time. Here, we demonstrateefficient “sequential” gene regulation using Cre-and FLP-expressing recombinant adenovirus(rAd) in two different monitor cell lines, forregulation of one gene (OFF-ON-OFF) and fortwo genes (ON-OFF and OFF-ON, independ-ently). Generally, serial use of Cre-and FLP-expressing rAd tends to cause significant cyto-toxicity, but we here described optimum dose ofthe rAds for serial regulation. We also estab-

lished an efficient method of rAd infection tomouse ES cell lines after removing feeder cells,showing that this system is useful for removalof FRT-flanked drug-resistance gene cassettefrom recombinant ES cells prior to introductionof ES cells into blastocytes for chimeric miceproduction. Because our sequential gene-regulation system offers efficient purpose-generegulation and strict OFF-regulation, it is poten-tially valuable for elucidating not only novelgene functions using cDNA microarray analysisbut also for “gene switching” in developmentand regeneration research.

4. The murine homolog of SALL4, a causativegene in Okihiro syndrome, is essential forembryonic stem cell proliferation, and co-operates with Sall1 in anorectal, heart,brain, and kidney development.

Masayo Sakaki-Yumoto4, Chiyoko Kobayashi4,Akira Sato5, Sayoko Fujimura4, Yuko Matsu-moto5, Minoru Takasato6, Tatsuhiko Kodama7,Hiroyuki Aburatani7 , Makoto Asashima6 ,Nobuaki Yoshida and Ryuichi Nishinaka-mura4,5,8: 4Division of Integrative Cell Biology,Institute of Molecular Embryology and Genet-ics, Kumamoto University, 5Division of StemCell Regulation, The Institute of Medical Sci-ence, The University of Tokyo, 6GraduateSchool of Sciences, The University of Tokyo, 7

Research Center for Advanced Science andTechnology, The University of Tokyo, 8

PRESTO, JST,

Mutations in SALL4 , the human homolog ofthe Drosophila homeotic gene spalt (sal) , causethe autosomal dominant disorder known as Oki-hiro syndrome. In this study, we show that atargeted null mutation in the mouse Sall4 geneleads to lethality during peri-implantation.Growth of the inner cell mass from the knock-out blastocysts was reduced, and Sall4-null em-bryonic stem (ES) cells proliferated poorly withno aberrant differentiation. Furthermore, wedemonstrated that anorectal and heart anoma-lies in Okihiro syndrome are caused by Sall4haploinsufficiency and that Sall4/Sall1 heterozy-gotes exhibited an increased incidence of anorec-tal and heart anomalies, exencephaly and kid-ney agenesis. Sall4 and Sall1 formed het-erodimers, and a truncated Sall1 caused mislo-calization of Sall4 in the heterochromatin; thus,some symptoms of Townes-Brocks syndromecaused by SALL1 truncations could result fromSALL4 inhibition.

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5. Genetic marking of hematopoietic stemand endothelial cells: Identification of theTmtsp gene encoding a novel cell-surfaceprotein with the thrombospondin-1 do-main.

Shin-ichiro Takayanagi9, Takashi Hiroyama10,Satoshi Yamazaki9, Tomoko Nakajima11, YoheiMorita9, Joichi Usui9, Koji Eto9, Tsutomu Mo-tohashi12, Kensuke Shiomi13, Kazuko Keino-Masu13, Masayuki Masu13, Yuichi Oike14, ShigeoMori15, Nobuaki Yoshida, Atsushi Iwama16, andHiromitsu Nakauchi9: 9Laboratory of Stem CellTherapy, the Center for Experimental Medi-cine, Institute of Medical Science, University ofTokyo, 10Cell Engineering Division, RIKENBioResource Center, 11Division of Cancer CellResearch, Department of Cancer Biology Insti-tute of Medical Science, University of Tokyo, 12

Department of Tissue and Organ Development,Regeneration and Advanced Medical Science,Gifu University Graduate School of Medicine,13Department of Molecular Neurobiology, Insti-tute of Basic Medical Sciences, GraduateSchool of Comprehensive Human Sciences,University of Tsukuba, 14Department of CellDifferentiation, the Sakaguchi Laboratory,School of Medicine, Keio University, 15Depart-ment of Pathology, Teikyo University School ofMedicine, 16Department of Cellular and Mo-lecular Medicine, Graduate School of Medicine,Chiba University.

Using an in silico database search, we identi-fied a novel gene encoding a cell surface mole-cule with a thrombospondin domain, and desig-nated the gene as transmembrane molecule withthrombospondin module (Tmtsp). Expressionprofiling of Tmtsp using specific monoclonal an-tibodies and Venus, a variant of yellow fluores-cent protein knock-in mice in the Tmtsp locus,demonstrated its specific expression in hema-topoietic and endothelial cells. In lymphohema-topoietic cells, Tmtsp was predominantly ex-pressed in hematopoietic stem and progenitorcells, and the level of expression gradually de-clined as the cells differentiated. Venus expres-sion faithfully traced the expression of Tmtsp ,and the level of Venus expression correlatedwell to the in vitro hematopoietic activity aswell as the in vivo bone marrow repopulatingcapacity. Notably, Venus expression marked thedevelopment of definitive hematopoiesis in boththe extraembryonic yolk sac and the intraembry-onic aorta-gonadmesonephros (AGM) regionand, in combination with CD41, strikingly pro-moted the enrichment of developing progenitorsin the CD41＋Venushigh fraction at embryonicday 10.5 (E10.5). In this context, Tmtsp is a novel

marker gene for primitive hematopoietic cellsand endothelial cells, and TmtspVenus/＋ micewould serve as a valuable mouse model for theanalysis of both embryonic and adult hema-topoiesis, as well as for vascular biology.

6. An Establishment of Mouse EmbryonicStem Cells Derived from (C57BL/6×129) F1 Blastocysts for High-throughput GeneTargeting.

Taeko Ichise, Reiko Sakamoto, Hirotake Ichise,Nobuaki Yoshida.

The whole genome sequence of a widely usedmouse strain, C57BL/6J(B6), has been completedand almost all clones of RPCI23-BAC B6 mousegenomic DNA library are mapped on the wholegenome contig of the mouse. The BAC clonesharboring genes of interest and their DNA se-quences can be obtained speedily and inexpen-sively. Moreover, recombinase-mediated modifi-cation of plasmid DNAs, the so-called “recom-beneering,” has been established and utilized forhigh-throughput DNA engineering. Hence, con-struction of a gene-targeting vector from a B6BAC clone is a preferable and time-savingmethod. However, B6-derived BAC insert DNAsare non-isogenic to genomic DNAs of widelyused ES cell lines derived from 129 substrains,though isogenic DNAs are preferred for con-struction of targeting vectors because mousestrain differences of DNA sequences betweentargeting vectors and genomic DNAs of ES cellscan reduce frequencies of homologous recombi-nation.

To utilize B6 BAC clones for gene targetingwithout the “non-isogenic DNA” problem, weestablished a new ES cell line derived from (B6×129) F1 blastocysts. The ES cells can contributeefficiently to both somatic and germ cells in chi-meric mice as determined by coat color andgerm-line transmission. We also obtained geneti-cally modified ES cells by using the ES cell lineand a B6 BAC-based targeting vector. Our newES cell line will be helpful for BAC-assisted high-throughput genetic modification of the mouse.

7. Generation of Transgenic Mice ExpressingTamoxifen-inducible Cre Recombinase forTemporal Genetic Modification In Vivo .

Akiko Hori, Hirotake Ichise, Taeko Ichise, SakiKondoh17, Yumi Kanegae17, Izumu Saito17,Nobuaki Yoshida: 17Laboratory of MolecularGenetics, IMSUT

Cre/loxP recombination system enables us toperform conditional genetic modification in the

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mouse; loxP-flanked sequences are removedfrom modified loci in the tissue-specific manner,based on tissue-specific promoter-driven Cre ex-pression. However, temporal genetic modifica-tion, which is often needed to circumvent unex-pected lethal phenotype of the genetically engi-neered mice, cannot be regulated by tissue-specific promoters used for transgenesis.

To perform the temporal genetic modification,we generated transgenic mice expressingtamoxifen-inducible Cre recombinase via ES cell-mediated transgenesis. We found that admini-stration of hydroxytamoxifen induced Cre-mediated recombination in the ES cells express-ing the tamoxifen-inducible Cre recombinasegene. We are now determining whethertamoxifen-dependent Cre/loxP recombinationcan be induced in the transgenic mice.

8. An Improved Transgenic Mouse Line Ex-pressing Temperature-Sensitive SV40 tsA58 T Antigen Based on Cre/loxP Recombi-nation System

Takashi Yamaguchi, Hirotake Ichise, OsamuIwata, Akiko Hori, Masaru Nakamura, TaekoIchise, Nobuaki Yoshida

Transgenically expressed temperature-sensitive SV40 large T antigen (tsA58 T Ag) hasbeen utilized for immortalization and mainte-nance of terminally differentiated cells. How-ever, its oncogenic activities in vivo lead to hy-perproliferation of T Ag-expressing cells, result-ing in unwanted lethal phenotypes. In order tocircumvent this problem, we established a trans-genic mouse line, termed “T26,” expressing tsA58 T Ag in Cre-loxP recombination-dependentmanner. T26 mice express β-geo in a variety oftissues throughout their development, whereasT26 mice harboring Cre transgene express tsA58T Ag instead of β-geo. tsA58 T Ag-expressingendothelial cells of T26/Tie2-Cre double trans-genic mice are obtained and maintained by asimple method for at least 1-2 months withoutalteration of endogenous cell characteristics.These results demonstrate that our T26 micemodel, in combination with Cre transgenic micemodel, is a useful tool for the simple and con-venient isolation and maintenance of somaticcells, providing an accessible model system forcell biology.

9. An Analysis of the Role of VEGF-C Signalin Blood Vascular Development in theMouse

Seiji Shiozawa, Hirotake Ichise, Akiko Hori,Taeko Ichise, Nobuaki Yoshida

Vascular endothelial growth factor receptor-3(VEGFR-3), a receptor for vascular endothelialgrowth factors (VEGF)-C and -D, is predomi-nantly expressed not only on lymphatic endo-thelial cells but on vascular endothelial cells inearly embryonic development . VEGFR-3-deficient mice fail to form large blood vesselsand die before the emergence of lymphatic ves-sel, suggesting that VEGFR-3 plays a critical rolein blood vascular development. However,knockout mice for either VEGF-C or -D exhibitno overt blood vascular phenotype. The role ofVEGFR-3 signaling in blood vascular develop-ment is still unknown.

In order to analyze the function of VEGFR-3signaling during embryonic vascular develop-ment in vivo, we established a transgenic ES cellline and a transgenic mouse line expressingVEGF-C in Cre-loxP recombination-dependentmanner. We found that endothelial proliferationand enlargement of the blood vasculature wereinduced in embryoid bodies and yolk sacs ofearly post-implantation embryos overexpressingVEGF-C. We are performing detail analyses tounderstand the role of VEGFR-3 signaling in theblood vasculature.

10. A Genetic Analysis of the Generation andSeparation of Blood and Lymphatic Vascu-lature in the Mouse

Hirotake Ichise, Taeko Ichise, Osamu Otani18,Nobuaki Yoshida: 18Department of Anatomy,Graduate School of Medicine and Pharmaceu-tical Sciences for Research, University ofToyama,

Blood vascular and lymphatic endothelial cellsshare a common developmental origin, as wellas molecular mechanisms of proliferation, motil-ity, and cell survival. The differentiation of lym-phatic endothelial cells from a subset of venousendothelial cells is initiated by the transcriptionfactor Prox1, followed by VEGF-C-induced cen-trifugal sprouting and budding of Prox-1＋ en-dothelial cells. Recent studies have shown thatbone marrow-derived circulating cells express-ing Syk and SLP-76 participate in the formationand maintenance of lymphatic vasculature sepa-rately from the blood vasculature. However, themechanism of the vascular separation is stilllargely unknown.

We identified a spontaneous mutant mousestrain exhibiting chylous ascites and blood-lymphatic shunt and found a candidate gene forthe mutation by positional candidate cloning.We are now investigating the importance ofcells for coordinating the generation and separa-tion of blood and lymphatic vasculature.

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11. Function of polypyrimidine tract bindingprotein in mouse embryonic stem cells

Satona Ohno, Hidehiko Tamura, TakashiOsaka, Mitsuharu Sato and Nobuaki Yoshida

Polypyrimidine tract binding protein (PTB),originally identified as an RNA binding proteinin hnRNP complex, was also known to be in-volved in the regulation of transcription andtranslation. In the analysis of Rex-1 gene pro-moter, we found PTB bind to polypyrimidinesequence within the promoter.

To study the role of PTB for embryonic stem(ES) cell function, we established PTB deficientES cell lines by gene targeting. The PTB defi-cient cells form normal colonies, but doublethymidine block analysis revealed that G2/Mprogression was delayed in these cells. The ex-pression levels of cyclinA or cyclinB were notaffected by PTB deficiency. It is still unclearwhether PTB directly regulates cell cycle ma-chinery.

12. PTB negatively regulates brain PTB ex-pression

Hidehiko Tamura, Mitsuharu Sato andNobuaki Yoshida

During the course of analysis in PTB deficientES cells, we found one of the PTB family mem-bers, brain PTB (brPTB/PTBP2) was up regu-lated in the absence of PTB. To test whether PTBsuppresses brPTB promoter, we generatedbrPTB-luciferase construct and cotransfectedwith PTB expression vector into PTB deficientES cells. The promoter activity was suppressedto 50％ of control when PTB expression vectorwas cotransfected. In addition, endogenousbrPTB expression was also down regulated byretrovirus infection expressing PTB in PTB defi-cient ES cells. These results explain why PTBand brPTB are exclusively expressed in mousedevelopment.

13. Analysis of Fbxl10 in mouse embryonicstem cells

Tsuyoshi Fukuda , Mitsuharu Sato andNobuaki Yoshida

Recently, one of the f-box protein family, Fbxl10, was shown to have histone demethylase ac-tivity. To examine the role of Fbxl10 for the EScell function, we analyzed the expression of Fbxl10 mRNA in mouse ES cells. Two bands weredetected in Northern blotting analysis when fulllength Fbxl10 cDNA or 3’ half of the cDNA

fragment was used as a probe, but with 5’ halfof the cDNA probe, only upper band was hy-bridized. On database, we noticed Fbxl10 has aninternal promoter located between exon 12 andexon 13 of full length Fbxl10. The shorter tran-script, which may corresponds to the lowerband detected in ES cells, does not encode JmjCdomain that is responsible for the histone de-methylase activity. We postulated that the shortform of Fbxl10 modulates histone demethylaseactivity of Fbxl10. To test this idea, we designedshRNA targeting only short form of Fbxl10 andsuccessfully down regulated the transcript. Weare analyzing the effect of the down regulationfor ES cell pluripotency and the methylationstate of the histone.

14. Activation of early retrotransposon (ETn)in mouse embryonic stem cells

Mio Furutani, Reiko Sakamoto, NobuakiYoshida and Mitsuharu Sato

We isolated early retrotransposon (ETn) as anES or EC (embryonal carcinoma) cell specificgene by differential hybridization. ETn is anLTR type retrotransposon, and over 900 ETnLTRs are distributed throughout the mousegenome. Although ETn does not encode for anyprotein, it is abundantly transcribed in undiffer-entiated ES or EC cells. We are interested intheir activation mechanism, and trying to knowwhether the transcription from such a repetitivesequence contributes to ES cell pluripotency.Our idea is that the activation of repetitive tran-scription units within the genome is essential formaintaining ES cell-specific chromatin structureand gene expressions. In order to understandthe activation mechanism of ETn, we have dis-sected ETn LTR and identified the regulatory se-quence that is essential for LTR transcriptionalactivity. Within the sequence, undifferentiatedcell-specific DNA binding activity is observed.To identify the factor responsible for the ES cell-specific ETn transcription, we have performedprotein purification and sought the DNA bind-ing activity seen in the regulatory sequence.From the reasons listed below, we supposedBORIS (brother of regulator of imprinted sites)as the regulator of ES cell-specific ETn expres-sion.1) There is the CCCTC motif in the regulatory

sequence.2) BORIS is expressed in ES cells and down

regulated during retinoic acid induced differ-entiation.

3) Proteins recovered from 75-100 kDa region onSDS-PAGE give the DNA binding activity onthe regulatory sequence.

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4) The shifted band observed with the regula-tory sequence in EMSA is competed by thenon-labeled FII sequence (which is known tobind BORIS).

5) When BORIS is over expressed in NIH 3T3,

the DNA binding activity found in ES cellsappears in nuclear extract.

6) Transcriptional activity from LTR is up regu-lated by BORIS expression in luciferase assay.

Publications

1. Seiji Kawa, Chizuru Ito, Yoshiro Toyama, Ma-miko Maekawa, Tohru Tezuka, TakahisaNakamura, Takanobu Nakazawa, KazumasaYokoyama, Nobuaki Yoshida, Kiyotaka Toshi-mori, and Tadashi Yamamoto.: Azoospermiain mice with targeted disruption of the Brek/Lmtk2 (brain-enriched kinase/lemur tyrosinekinase 2) gene. Proc. Natl. Acad. Sci., USA,103: 19344-19349, 2006

2. Saki Kondo,Yuzuka Takahashi,Seiji Shiozawa,Hirotake Ichise, Nobuaki Yoshida, YumiKanegae, and Izumi Saito.: Efficient sequentialgene regulation via FLP- and Cre-recombinaseusing adenovirus vector in mammalian cellsincluding mouse ES cells. Microbiol. Immu-nol., 50: 831-843, 2006

3. Masayo Sakaki-Yumoto, Chiyoko Kobayashi,Akira Sato, Sayoko Fujimura, Yuko Matsu-moto, Minoru Takasato, Tatsuhiko Kodama,

Hiroyuki Aburatani , Makoto Asashima ,Nobuaki Yoshida and Ryuichi Nishinaka-mura.: The murine homolog of SALL4, acausative gene in Okihiro syndrome, is essen-tial for embryonic stem cell proliferation, andcooperates with Sall1 in anorectal, heart,brain, and kidney development. Development,133: 3005-3013, 2006

4. Shin-ichiro Takayanagi, Takashi Hiroyama,Satoshi Yamazaki, Tomoko Nakajima, YoheiMorita, Joichi Usui, Koji Eto, Tsutomu Moto-hashi, Kensuke Shiomi, Kazuko Keino-Masu,Masayuki Masu, Yuichi Oike, Shigeo Mori,Nobuaki Yoshida, Atsushi Iwama, and Hiro-mitsu Nakauchi.: Genetic marking of hema-topoietic stem and endothelial cells: identifica-tion of the Tmtsp gene encoding a novel cellsurface protein with the thrombospondin-1domain. Blood, 107: 4317-4325, 2006

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Stem cells are generally defined as clonogenic cells capable of both self-renewaland multilineage differentiation. Because of these unique properties, stem cells of-fer the novel and exciting possibility of organ reconstitution in place of trans-planted or artificial organs in the treatment of organ failure. In addition, stem cellsare considered as ideal target cells for gene/cell therapy. The goal of this labora-tory is to provide new insights into stem cell biology as well as approaches totherapeutic intervention for various intractable diseases.

1. The mechanism of hematopoietic stem cellself-renewal and commitment.

a) Cytokine signals modulated via lipid raftsmimic niche signals and induce hiberna-tion in hematopoietic stem cells

Yamazaki S, Iwama A, Takayanagi S, MoritaY, Eto K, Ema H, Nakauchi H.

Hematopoietic stem cells (HSCs) reside in thebone marrow (BM) niche in a non-cycling stateand enter the cell cycle at long intervals. How-ever, little is known about inter- and intra-cellular signaling mechanisms underlying thisunique property of HSCs. In this study, weshow that lipid raft clustering is a key event inthe regulation of HSC dormancy. Freshly iso-lated HSCs from the BM niche lack lipid raftclustering, exhibit repression of the AKT-FOXOsignaling pathway, and express abundant p57Kip2

cyclin-dependent kinase inhibitor. Lipid raftclustering induced by cytokines is essential forHSC re-entry into the cell cycle. Conversely, in-hibition of lipid raft clustering caused sustainednuclear accumulation of FOXO transcription fac-

tors and induced HSC hibernation ex vivo. Thesedata establish a critical role for lipid rafts inregulating the cell cycle, the survival, and theentry into apoptosis of HSCs and uncover astriking similarity in HSC hibernation and C. ele-gans dauer formation.

b) Non-side-population hematopoietic stemcells in the bone marrow

Morita Y, Ema H, Yamazaki S, Nakauchi H.

Most hematopoietic stem cells (HSCs) are as-sumed to reside in the so-called side population(SP) in adult mouse bone marrow (BM). We re-port the coexistence of non-SP HSCs that do notsignificantly differ from SP HSCs in numbers,capacities, and cell-cycle states. When stainedwith Hoechst 33342 dye, the CD34(－/low) c-Kit(＋)Sca-1(＋)lineage marker(－) (CD34(－)KSL)cell population, highly enriched in mouse HSCs,was almost equally divided into the SP and themain population (MP) that represents non-SPcells. Competitive repopulation assays with sin-gle or 30 SP- or MP-CD34(－)KSL cells foundsimilar degrees of repopulating activity and fre-

Center for Experimental Medicine

Laboratory of Stem Cell Therapy高次機能（幹細胞治療）研究分野

Professor Hiromitsu Nakauchi M.D., Ph.D.Research Associate Koji Eto M.D., Ph.D.Research Associate Makoto Otsu M.D., Ph.D.Research Associate Akihide Kamiya Ph.D.

教 授 医学博士 中 内 啓 光助 手 医学博士 江 藤 浩 之助 手 医学博士 大 津 真助 手 理学博士 紙 谷 聡 英

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quencies of repopulating cells for these popula-tions. Secondary transplantation detected self-renewal capacity in both populations. SP analy-sis of BM cells from primary recipient mice sug-gested that the SP and MP phenotypes are inter-convertible. Cell-cycle analyses revealed that CD34(－)KSL cells were in a quiescent state andshowed uniform cell-cycle kinetics, regardless ofwhether they were in the SP or MP. Bcrp-1 ex-pression was similarly detected in SP- and MP-CD34(－)KSL cells, suggesting that the SP phe-notype is regulated not only by Bcrp-1, but alsoby other factors. The SP phenotype does notspecify all HSCs; its identity with stem cell func-tion thus is unlikely.

c) Differential impact of Ink4a and Arf on he-matopoietic stem cells and their bone mar-row microenvironment in Bmi1-deficientmice

Oguro H, Iwama A1, Morita Y, Kamijo T1, vanLohuizen M2, Nakauchi H: 1Graduate School ofMedicine, Chiba University, 2The NetherlandsCancer Institute

The polycomb group (PcG) protein Bmi1plays an essential role in the self-renewal of he-matopoietic and neural stem cells. Derepressionof the Ink4a/Arf gene locus has been largely at-tributed to Bmi1-deficient phenotypes in thenervous system. However, its role in hema-topoietic stem cell (HSC) self-renewal remainedundetermined. In this study, we show thatderepressed p16(Ink4a) and p19(Arf) in Bmi1-deficient mice were tightly associated with aloss of self-renewing HSCs. The deletion of bothInk4a and Arf genes substantially restored theself-renewal capacity of Bmi1(－/－) HSCs.Thus, Bmi1 regulates HSCs by acting as a criti-cal failsafe against the p16(Ink4a)- and p19(Arf)-dependent premature loss of HSCs. We furtheridentified a novel role for Bmi1 in the organiza-tion of a functional bone marrow (BM) microen-vironment. The BM microenvironment in Bmi1(－/－) mice appeared severely defective in sup-porting hematopoiesis. The deletion of both Ink4a and Arf genes did not considerably restore theimpaired BM microenvironment, leading to asustained postnatal HSC depletion in Bmi1(－/－)Ink4a-Arf(－/－) mice. Our findings unveil adifferential role of derepressed Ink4a and Arf onHSCs and their BM microenvironment in Bmi1-deficient mice. Collectively, Bmi1 regulates self-renewing HSCs in both cell-autonomous andnonautonomous manners.

d) Genetic marking of hematopoietic stemand endothelial cells: identification of the

Tmtsp gene encoding a novel cell surfaceprotein with the thrombospondin-1 do-main.

Takayanagi S, Hiroyama T1, Yamazaki S,Nakajima T, Morita Y, Usui J, Eto K, Moto-hashi T2, Shiomi K3, Keino-Masu K3, Masu M3,Oike Y4, Mori S, Yoshida N, Iwama A5,Nakauchi H: 1RIKENBRC, 2Graduate Schoolof Medicine, Gifu University, 3 GraduateSchool of Comprehensive Human Sciences,University of Tsukuba, 4School of Medicine,Keio University, 5Graduate School of Medicine,Chiba University

Using an in silico database search, we identi-fied a novel gene encoding a cell surface mole-cule with a thrombospondin domain, and desig-nated the gene as transmembrane molecule withthrombospondin module (Tmtsp). Expressionprofiling of Tmtsp using specific monoclonal an-tibodies and Venus, a variant of yellow fluores-cent protein knock-in mice in the Tmtsp locus,demonstrated its specific expression in hema-topoietic and endothelial cells. In lymphohema-topoietic cells, Tmtsp was predominantly ex-pressed in hematopoietic stem and progenitorcells, and the level of expression gradually de-clined as the cells differentiated. Venus expres-sion faithfully traced the expression of Tmtsp,and the level of Venus expression correlatedwell to the in vitro hematopoietic activity aswell as the in vivo bone marrow repopulatingcapacity. Notably, Venus expression marked thedevelopment of definitive hematopoiesis in boththe extraembryonic yolk sac and the intraembry-onic aorta-gonad-mesonephros (AGM) regionand, in combination with CD41, strikingly pro-moted the enrichment of developing progenitorsin the CD41(＋)Venus(high) fraction at embry-onic day 10.5 (E10.5). In this context, Tmtsp is anovel marker gene for primitive hematopoieticcells and endothelial cells, and Tmtsp(Venus/)(＋) mice would serve as a valuable mousemodel for the analysis of both embryonic andadult hematopoiesis, as well as for vascular biol-ogy.

2. Stem/progenitor cells in hepato-biliary sys-tem

a) Side population purified from hepatocellu-lar carcinoma cells harbors cancer stemcell-like properties.

Chiba T1, Kita K2, Zheng YW2, Yokosuka O2,Saisho H2, Iwama A1, Nakauchi H, TaniguchiH2: 1Graduate School of Medicine, Chiba Uni-versity, 2Graduate School of Medicine Yoko-

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hama City University

Recent advances in stem cell biology enableus to identify cancer stem cells in solid tumorsas well as putative stem cells in normal solid or-gans. In this study, we applied side population(SP) cell analysis and sorting to established he-patocellular carcinoma (HCC) cell lines to detectsubpopulations that function as cancer stemcells and to elucidate their roles in tumorigene-sis. Among four cell lines analyzed, SP cellswere detected in Huh7 (0.25％) and PLC/PRF/5cells (0.80％), but not in HepG2 and Huh6 cells.SP cells demonstrated high proliferative poten-tial and anti-apoptotic properties compared withthose of non-SP cells. Immunocytochemistry ex-amination showed that SP fractions contain alarge number of cells presenting characteristicsof both hepatocyte and cholangiocyte lineages.

Non-obese diabetic/severe combined immu-nodeficiency (NOD/SCID) xenograft transplantexperiments showed that only 1×103 SP cellswere sufficient for tumor formation, whereas aninjection of 1×106 non-SP cells did not initiatetumors. Re-analysis of SP cell-derived tumorsshowed that SP cells generated both SP and non-SP cells and tumor-initiating potential wasmaintained only in SP cells in serial transplanta-tion. Microarray analysis discriminated a differ-ential gene expression profile between SP andnon-SP cells, and several so-called “stemnessgenes” were upregulated in SP cells in HCCcells. In conclusion, we propose that a minoritypopulation, detected as SP cells in HCC cells,possess extreme tumorigenic potential and pro-vide heterogeneity to the cancer stem cell sys-tem characterized by distinct hierarchy.

Publications

Ema, H., Morita, Y., Yamazaki, S., Matsubara,A., Seita, Y., Tadokoro, Y., Takano, H., Takaki,S. and Nakauchi, H. Adult mouse hema-topoietic stem cells: Purification and single-cell assays. Nature Protocols . 1: 2979-2987,2006.

Hamanaka S, Nabekura T, Otsu M, Yoshida H,Nagata M, Usui J, Takahashi S, Nagasawa T,Nakauchi H, Onodera M. Stable TransgeneExpression in Mice Generated from Retrovi-rally Transduced Embryonic Stem Cells. MolTher . in press Epub Dec 19. 2006.

Negishi M, Saraya A, Miyagi S, Nagao K, Ina-gaki Y, Nishikawa M, Tajima S, Koseki H,Tsuda H, Takasaki Y, Nakauchi H, Iawama A.Bmi1 cooperates with Dnmt1-assciated protein1 in gene silencing. Biochem Biophys Res Com-mun . in press Epub Dec 29. 2006.

Suzuki A, Ivandini TA, Kamiya A, Nomura S,Yamanuki M, Matsumoto K, Fujishima A,Einaga Y. Direct electrochemical detection ofsodium azide in physiological salinebuffersusing highly boron-doped diamond elec-trodes. Sensors and Actuators B . in press

Yamazaki S, Iwama A, Takayanagi S-I, MoritaY, Eto K, Ema H, and Nakauchi H. Cytokinesignals modulated via lipid rafts mimic nichesignals and induce hibernation in hematopoie-tic stem cells. EMBO J 25 : 3515-3523, 2006.

Morita Y. Ema H. Yamazaki S.and Nakauchi H.Non-side-population hematopoietic stem cellsin mouse bone marrow. Blood 108 : 2850-2856,2006.

Nabekura T, Otsu M, Nagasawa T, Nakauchi H,Onodera M. Potent Vaccine Therapy withDendritic Cells Genetically Modified by the

Gene-Silencing-Resistant Retroviral VectorGCDNsap. Mol Ther. 13: 301-309, 2006.

Furuta C, Ema H, Takayanagi S-i, Ogaeri T,Okamura D, Masui Y and Nakauchi H. Dis-cordant developmental waves of angioblastsand hemangioblasts in the early gastrulatingmouse embryo. Development 133 : 2771-2779,2006.

Suzuki E, Tsutsumi A, Goto D, Matsumoto I, ItoS, Otsu M, Onodera M, Takahashi S, Sato Y,Sumida T. Gene transduction of tristetraprolinor its active domain reduces TNF-alpha pro-duction by Jurkat T cells. Int J Mol Med . 17:801-809, 2006.

Thrasher AJ, Gaspar HB, Baum C, Modlich U,Schambach A, Candotti F, Otsu M, SorrentinoB, Scobie L, Cameron E, Blyth K, Neil J, AbinaSH, Cavazzana-Calvo M, Fischer A. Genetherapy: X-SCID transgene leukaemogenicity.Nature . 443: E5-6; discussion E6-7, 2006.

Soriani A, Moran B, de Virgilio M, Kawakami T,Altman A, Lowell C, Eto K, Shattil SJ. A rolefor PKCq in outside-in aIIbb3 signaling. JThromb Haemost. 4: 648-655, 2006.

Oki T, Kitaura J, Eto K, Lu Y, Maeda-YamamotoM, Inagaki N, Nagai H, Yamanashi Y, Naka-jima H, Kumagai H, Kitamura T. IntegrinaIIbβ3 induces the adhesion and activaton ofmast cells through interaction with fibrinogen.J Immunol . 176: 52-60, 2006.

Takayanagi S-i, Hiroyama T, Yamazaki S, Naka-jima T, Morita Y, Usui J, Eto K, Motohashi T,Shiomi K, Keino-Masu K, Oike Y, Mori S,Yoshida N, Iwama A, Nakauchi H. Geneticmarking of hematopoietic stem and endothe-lial cells: identification of the Tmtsp gene en-

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coding a novel cell surface protein with thethrombospondin-1 domain. Blood . 107: 4317-4325, 2006.

Kamiya A, Gonzalez FJ, Nakauchi H. Identifica-tion and differentiation of hepatic stem cellsduring liver development. Front Biosci . 11:1302-1310. Review, 2006.

Choudhury AR, Ju Z, Djojosubroto MW,Schienke A, Lechel A, Schaetzlein S, Jiang H,Stepczynska A, Wang C, Buer J, Lee HW, vonZglinicki T, Ganser A, Schirmacher P,Nakauchi H, Rudolph KL. Cdkn1a deletionimproves stem cell function and lifespan ofmice with dysfunctional telomeres without ac-celerating cancer formation. Nature Genet . 39:99-105, 2007 Epub, 2006

Oguro H, Iwama A, Morita Y, Kamijo T, van

Lohuizen M, Nakauchi H. Differential impactof Ink4a and Arf on hematopoietic stem cellsand their bone marrow microenvironment inBmi1-deficient mice. J Exp Med . 203: 2247-53,2006

Sakaguchi T, Nishimoto M, Miyagi S, Iwama A,Morita Y, Iwamori N, Nakauchi H, KiyonariH, Muramatsu M, Okuda A. Putative“stemness” gene jam-B is not required formaintenance of stem cell state in embryonic,neural, or hematopoietic stem cells. Mol CellBiol . 26: 6557-70, 2006

Chiba T, Kita K, Zheng YW, Yokosuka O, SaishoH, Iwama A, Nakauchi H, Taniguchi H. Sidepopulation purified from hepatocellular carci-noma cells harbors cancer stem cell-like prop-erties. Hepatology . 44: 240-51, 2006

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