千葉大学バイオメディカル研究センター
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研究概要

バイオメデイカル研究センターでは、疾患モデルマウスを用いて次のようなテーマで研究を行っています。


1.

神経堤細胞の異常に起因する疾患の分子遺伝学的解析

 神経堤細胞は胎生期に神経管背側より発生し、全身を移動し腸管神経細胞、交感神経系、皮膚メラニン色素細胞、副腎髄質などへと分化します。これらの細胞の発生、分化異常により、ヒルシュスプルング病およびその類縁疾患、神経芽細胞腫などの疾患が発症します。私たちは神経堤細胞に特異的に発現する遺伝子Ncxのノックアウトマウスを作製しヒルシュスプルング病類縁疾患のモデルマウスであることを報告してきました。現在神経堤細胞で発現する遺伝子に注目し、そのノックアウトマウスの解析から神経堤細胞の異常に起因する疾患(Neurocristopathy)のなりたちについて明らかにしようとしています。

2. 腸管神経系と腸管免疫系の相互作用

消化管においては腸内細菌と免疫系の相互作用により消化管免疫系細胞の分化、恒常性が制御されていることが最近の消化管免疫の研究より明らかになっています。一方消化管には全身の神経細胞の約半数が存在するといわれ第二の脳とも言われています。私たちは腸管神経と腸管免疫系および腸管バリア機構に着目し腸管神経系に異常をきたす遺伝子組み換えマウスを用いて腸管神経が腸管免疫系、腸内フローラおよび腸管上皮バリア機構に及ぼす相互作用についての解析を行っています。

3.

遺伝子組み換えマウスを用いた細胞増殖・分化の分子機構の解析

 個体発生胎生期における細胞増殖制御のしくみ、肺胞形成の分子機構などについて遺伝子組み換えマウスを用いて分子生物学的、細胞生物学的解析を行っています。

4.

その他、他大学・研究所との共同研究により各種疾患モデルマウスの作製・解析を行っています。

主な発表論文

主な発表論文(2001年以降) (*は当研究室で作製したマウスです)


2014
*2. Watanabe-Takano H, Takano K, Sakamoto A, Matsumoto K, Tokuhisa T, Endo T, Hatano M. DA-Raf-dependent inhibition of the Ras-ERK signaling pathway in type 2 alveolar epithelial cells controls alveolar formation. Proc Natl Acad Sci USA., 111, E2291-E2300, 2014
*1.
 Ikari J, Inamine A, Yamamoto T, Watanabe-Takano H, Yoshida N, Fujimura L, Taniguchi T, Sakamoto A, Hatano M, Tatsumi K, Tokuhisa T, Arima M. Plant homeodomain finger protein 11 promotes class switch recombination to IgE in murine activated B cells. Allergy 69, 223-230, 2014.
2013
6.  Takahashi W, Watanabe E, Fujimura L, Watanabe-Takano H, Yoshidome H, Swanson PE, Tokuhisa T, Oda S, Hatano M. Kinetics and protective role of autophagy in a mouse cecal ligation and puncture-induced sepsis. Crit Care 17, R160, 2013.
5. Kamoda H, Ohtori S, Ishikawa T, Miyagi M, Arai G, Suzuki M, Sakuma Y, Oikawa Y, Kubota G, Orita S, Eguchi Y, Yamashita M, Yamauchi K, Inoue G, Hatano M, Takahashi K. The effect of platelet-rich plasma on posterolateral lumbar fusion in a rat model. J Bone Joint Surg Am., 95, 1109-1116, 2013.
*4.  Ito C, Yamatoya K, Yoshida K, Fujimura L, Hatano M, Miyado K, Toshimori K. Integration of the mouse sperm fertilization-related protein equatorin into the acrosome during spermatogenesis as revealed by super-resolution and immunoelectron microscopy. Cell Tissue Res., 352, 739-750, 2013.
3. Okada K, Ueshima S, Kawao N, Yano M, Tamura Y, Tanaka M, Sakamoto A, Hatano M, Arima M, Miyata S, Nagai N, Tokuhisa T, Matsuo O. Lack of both α2-antiplasmin and plasminogen activator inhibitor type-1 induces high IgE production. Life Sci., 93, 89-95, 2013.
*2. Tsuruoka N, Arima M, Yoshida N, Okada S, Sakamoto A, Hatano M, Satake H, Arguni E, Wang JY, Yang JH, Nishikura K, Sekiya S, Shozu M, Tokuhisa T. ADAR1 protein induces adenosine-targeted DNA mutations in senescent Bcl6 gene-deficient cells. J Biol Chem., 288, 826-836, 2013.
*1.
Huang J, Li X, Kohno K, Hatano M, Tokuhisa T, Murray PJ, Brocker T, Tsuji M. Generation of tissue-specific H-2Kd transgenic mice for the study of K(d)-restricted malaria epitope-specific CD8+ T-cell responses in vivo. J Immunol Methods 387, 254-61, 2013.
2012
*1.
 Ohnuki H, Inoue H, Takemori N, Nakayama H, Sakaue T, Fukuda S, Miwa D, Nishiwaki E, Hatano M, Tokuhisa T, Endo Y, Nose M, Higashiyama S. BAZF, a novel component of cullin3-based E3 ligase complex, mediates VEGFR and Notch cross-signaling in angiogenesis. Blood 119, 2688-2698, 2012.

2011
*3. Seto T, Yoshitake M, Ogasawara T, Ikari J, Sakamoto A, Hatano M, Hirata H, Fukuda T, Kuriyama T, Tatsumi K, Tokuhisa T, Arima M. Bcl6 in pulmonary epithelium coordinately controls the expression of the CC-type chemokine genes and attenuates allergic airway inflammation. Clin Exp Allergy 41, 1568-1578, 2011.
*2. Yoshida N, Kitayama D, Arima M, Sakamoto A, Inamine A, Takano H, Hatano M, Koike T, Tokuhisa T. CXCR4 expression on activated B cells is downregulated by CD63 and IL21. J Immunol. 186, 2800-2808, 2011.
*1.
Ohtsuka H, Sakamoto A, Horigome S, Ichii H, Arima M, Hatano M, Okada S, Tokuhisa T. Bcl6 is required for the development of mouse CD4+ and CD8a+ dendritic cells. J Immunol. 186, 255-263, 2011.
2010
*2. Otaki JM, Hatano M, Matayoshi R, Tokuhisa T, Yamamoto H. The proto-oncogene BCL6 promotes survival of olfactory sensory neurons. Develop Neurobiol., 70, 434-435, 2010
*1.
Ohta, Y., Fujimura, L., Nishio, S., Arima, M., Sakamoto, A., Shimada, H., Ochiai, T., Tokuhisa, T. and Hatano, M. A kelch family protein Nd1-L functions as a metastasis suppressor in cancer cells via Rho family proteins mediated mechanism. Int. J. Oncol., 36, 427-434, 2010.
2009
 *2. Kato Y, Miyahara K, Hatano M, Hasegawa Y, Seki T, Frykman PK, Kusafuka J, Lane GJ, Yamataka A. Immature enteric neurons in Ncx/Hox11L.1 deficient intestinal neuronal dysplasia model mice. Pediatr Surg Int., 25, 961-965, 2009.
*1.
Fujimura L., Takano-Watanabe, H., Sato, Y., Tokuhisa, T. and Hatano, M. Prickle promotes neurite outgrowth via the Dishevelled dependent pathway in C1300 cells. Neuroscience Lett., 467, 6-10,2009.

2008
*5.
Wakashin, H., Hirose, K., Maezawa, Y., Kagami, S-I., Suto A., Watanabe, N., Saito, Y., Hatano, M., Tokuhisa, T., Iwakura, Y., Puccetti, P., Iwamoto, I. and Nakajima, H. IL-23 and Th17 cells enhance Th2 cell-mediated eosinophilic airway inflammation in mice.  Am. J. Resp. Crit. Care Med., 178, 1023-1032, 2008.
4.
Ouchida, R., Yamasaki, S., Hikida, M., Masuda, K., Kawamura, K., Wada, A., Mochizuki, S., Tagawa, M., Sakamoto, A., Hatano, M., Tokuhisa, T., Koseki, H., Saito, T., Kurosaki, T. and Wang, Ji-Y. A lysozomal protein negatively regulates surface T cell antigen receptor expression by promoting CD3zeta degradation. Immunity, 29, 33-43, 2008.
3.
Saito, T., Kitayama, D., Sakamoto, A., Tsuruoka, N., Arima, M., Hatano, M., Miyazaki, M. and Tokuhisa, T. A distinct role for IL-4 and IL-21 in their collaboration on proliferation and differentiation of activated B cells. Immunobiol., 213, 545-555, 2008.
2.
Kitayama, D., Sakamoto, A., Tsuruoka, N., Arima, M., Hatano, M., Miyazaki, M and Tokuhisa, T. A role for Bcl6 in sequential class switch recombination to IgE in B cells stimulated with IL-4 and IL-21. Mol. Immunol., 45, 1337-1345, 2008.
1.
Abe, R., Hirasawa, H., Oda, S., Shiga, H., Matsuda, K., Nakamura, M., Watanabe, E., Nakada, T., Hatano, M. and Tokuhisa, T. Up-regulation of interleukin-10 messenger RNA expression in peripheral leukocytes predicts poor outcome and diminished human leukocyte antigen-DR expression on monocytes in septic patients. J. Surg. Res., 147, 1-8, 2008.

2007
*5.
Tsuruoka, N., Arima, M., Arguni, E., Saito, T., Kitayama, D., Sakamoto, A., Hatano, M. and Tokuhisa, T. Bcl6 protects apoptosis of naive B cells stimulated with IL-21.  Immunol. Lett., 110, 145-151, 2007.
*4.
Kobayashi, H., Kusafuka, J., Lane, GJ., Yamataka, A., Satoh, K., Hayakawa, T., Kase, Y. and Hatano, M. The mechanism of intestinal motility in homozygous mutant Ncx/Hox11L.1-deficient mice--a model for intestinal neuronal dysplasia. J. Pediatr. Surg., 42, 2062-2066, 2007.
*3.
Ichii, H., Sakamoto, A., Arima, M., Hatano, M., Kuroda, Y. and Tokuhisa T. Bcl6 is essential for the generation of long-term memory CD4+ T cells. Int. Immunol., 19, 427-433, 2007.
2.
Kitahashi, M., Sato, Y., Fujimura, L., Ozeki, C., Arima, M., Sakamoto, A., Yamamoto, S., Tokuhisa, T. and Hatano, M. Identification of the consensus DNA sequence for Nczf-binding. DNA Cell Biol., 26, 395-401, 2007.
*1.
Aoki, T., Jusuf, A.A., Iitsuka, Y., Isono, K., Tokuhisa, T., and Hatano, M. Ncx (Enx, Hox11L.1) is required for the neuronal cell death in enteric ganglia of mice. J. Pediatr. Surg., 42, 1081-1088, 2007.

2006
6.
Yoshida, K., Sakamoto, A., Yamashita, K., Arguni, E., Horigome, S., Arima, M., Hatano, M., Seki, N., Ichikawa, T. and Tokuhisa, T. Bcl6 controls granzyme B expression in effector CD8+ T cells. Eur. J. Immunol., 36, 3146-3156, 2006.
*5.
Tamachi, T., Maezawa, Y., Ikeda, K., Kagami, S., Hatano, M., Seto, Y., Suto, A., Suzuki, K., Watanabe, N., Saito, Y., Tokuhisa, T., Iwamoto, I. and Nakajima, H. IL-25 enhances allergic airway inflammation by amplifying a TH2 cell-dependent pathway in mice. J. Allergy Clin. Immunol., 118, 606-614, 2006.
*4.
Takamori, Y., Matsudo, Y., Fujimura, L., Kang, M., Harada, Y., Moriya, H., Tokuhisa, T., and Hatano, M. Expression of the Nd1 gene is down-regulated by doxorubicin at post-transcriptional level. Int. J. Mol. Med., 18, 963-967, 2006
*3.
Maeda, Y., Fujimura, L., 0-Wang, J., Hatano, M., Sakamoto, A., Arima, M., Ebara, M., Ino, H., Yamashita, T., Saisho, H., and Tokuhisa, T. Role of Clast1 in differentiation of cerebellar granule cells. Mol. Brain Res., 1104, 18-26, 2006.
*2.
Matsudo, Y., Takamori, Y., Fujimura, L., Nishio, S., Sasagawa, K., Komuro, I., Tokuhisa, T., and Hatano, M. Overexpression of Nd1, a novel Ketch family protein, in the heart of transgenic mice protects against doxorubicin-induced cardiomyopathy. Transgenic Res., 15, 573-581, 2006.
1.
Arguni, E., Arima, M., Tsuruoka, N., Sakamoto, A., Hatano, M., and Tokuhisa, T. JunD/AP-1 and STAT3 are the major enhancer molecules for high Bcl6 expression in germinal center B cells. Int, Immunol., 18,1079-1089, 2006.

2005
9.
Nakada, T., Hirasawa, H., Oda, S., Shiga, H., Matsuda, K., Watanabe, E., Abe, R., Hatano, M., and Tokuhisa, T. Not toll-like receptor 4 nor CD14 polymorphysms but tumore necrosis factor and interleukin-10polymorphysms attenuate survival from sepsis in Japanese critically ill patients.  J. Surg. Res., 129: 322-328.
8.
Chandrasekar, B., Mummidi, S., Valente, A. J., Patel, D. N., Bailey, S. R., Freeman, G. L., Hatano, M., Tokuhisa, T., and Jensen, L. E. The pro-atherogenic cytokine interleukin-18 induces CXCL16 expression in rat aortic smooth muscle cells via MyD88, IRAK,TRAF6, c-Src, PI3K, Akt, JNK, and AP-1 signaling.  J. Biol. Chem., 280: 26263-26277.
7.
Ohkubo, Y., Arima, M., Arguni, E., Okada, S., Yamashita, K., Asari, S., Obata, S., Sakamoto, A., Hatano, M., O-Wang, J., Ebara, M., Saisho, H., and Tokuhisa, T. A role for c-Fos/AP-1 in B lymphocyte terminal differentiation.  J. Immunol., 174: 7703-7710.
6.
Kawakami, H., Tomita, M., Matsuda, T., Ohta, T., Tanaka, Y., Fujii, M., Hatano, M., Tokuhisa, T., and Mori, N. Transcriptional activation of survivin through the NF-kappaB pathway by human T-cell leukemia virus type I tax.  Int. J. Cancer, 115: 967-974.
*5.
Nishio, S., Hatano, M., Nagata,M., Horie,S., Koike,T., Tokuhisa,T., and Mochizuki, T. Pkd1 regulates immortalized proliferation of renal tubular epithelial cells through induction of P53 and activation of JNK.  J. Clin. Invest., 115: 910-918.
4.
Watanabe, E., Hirasawa, H., Oda, S., Matsuda, K., Hatano, M., and Tokuhisa, T. The Extremely High Interleukin-6 Blood Levels and Outcome in the Critically Ill Are Associated with Tumor Necrosis Factor- and Interleukin-1-Related Gene Polymorphisms. Crit. Care Med., 33: 89-97.
*3.
Asari, S., Sakamoto, A., Okada, S., Ohkubo, Y., Arima, M., Hatano, M., Kuroda, K., and Tokuhisa, T. Abnormal erythroid differentiation in neonatal Bcl6-deficient mice.  Exp. Hematol., 33: 26-34.
2.
Inoue, A., Kang, M., Fujimura, L., Takamori, Y., Sasagawa, K., Itoh, H., Tokuhisa, T., and Hatano, M. Overexpression of Nd1-S, a variant form of new kelch family protein, perturbs the cell cycle progression of fibroblasts. DNA Cell Biol., 24: 30-34.
1.
Yamashita, K., Sakamoto, A., Ohkubo, Y., Arima, M., Hatano, M., Kuroda, K., and Tokuhisa, T. C-fos overexpression in splenic B cells augments development of marginal zone B cells.  Mol. Immunol., 42: 617-625.

2004
*8.
Kuma, A., Hatano, M., Matsui, M., Yamamoto, A., Nakaya, H., Yoshimori, T., Ohsumi, Y., Tokuhisa, T., and Mizushima, N. Role of autophagy during the early neonatal starvation period. Nature, 432: 1032-1036.
7.
Mori, S., Sakamoto, A., Yamashita, K., Fujimura, L., Arima, M., Hatano, M., Miyazaki , M., and Tokuhisa, T. Effect of c-fos overexpression on development and proliferation of peritoneal B cells. Int. Immunol., 16: 1477-1486.
*6.
Fujimura, L., Matsudo , Y., Kang, M., Takamori, Y., Tokuhisa, T., and Hatano, M. Protective role of Nd1 in doxorubicin-induced cardiotoxicity. Cardiovasc. Res., 64: 315-321.
*5.
Takamori, M., Hatano, M., Arima, M., Sakamoto, A., Fujimura, L., Hartatik, T., Kuriyama, T., and Tokuhisa, T. BAZF is required for activation of naive CD4 T cells by TCR-triggering. Int. Immunol., 16 :1439-1449.
*4.
Yanai T, Kobayashi H, Yamataka A, Lane GJ, Miyano T, Hayakawa T, Satoh K, Kase Y, Hatano M. Acetylcholine-related bowel dysmotility in homozygous mutant NCX/HOX11L.1-deficient (NCX-/-) mice-evidence that acetylcholine is implicated in causing intestinal neuronal dysplasia. J Pediatr Surg. 39: 927-930.
3.
Asari, S., Okada, S., Ohkubo, Y., Sakamoto, A., Arima, M., Hatano, M., Kuroda, Y., and Tokuhisa, T. Early erythropoiesis in the bone marrow and spleen after stem cell transplantation. Transplantation, 78: 516-523.
2.
Kawamura, K., O-Wang, J., Ogawa, R., Bahar, R., Seimiya, M., Chiyo, M., Wada, A., Okada, S., Hatano, M., Tokuhisa, T., Kimura, H., Watanabe, S., Honda, I. , Sakiyama,S., and Tagawa, M. DNA polymerase ? is preferentially expressed in lymphoid tissues and upregulated in human cancers. Eur. J. Immunol., 34: 1322-1332.
1.
Seimiya, M., Wada, A., Kawamura, K., Sakamoto, A., Ohkubo, Y., Okada,S., Hatano, M., Tokuhisa, T., Watanabe, T., Saisho, H., Tagawa, M., and O-Wang, J. Impaired lymphocyte development and function in Clast5/Stra13/DEC1 transgenic mice . Int. J. Cancer, 109: 9-16.

2003
5.
Okada, S., Obata, S., Hatano, M., and Tokuhisa, T. Dominant negative effect of the c-fos family gene products on inducible nitric oxide synthase expression in macrophages. Int. Immunol., 15: 1275-1285.
4.
Honda, K., Arima, M., Cheng, G., Taki, S., Hirata, H., Eda, F., Fukushima , F., Yamaguchi, B., Hatano, M., Tokuhisa, T., and Fukuda, T. Prostaglandin D2 Reinforces Th2 Type Inflammatory Responses of Airways to Low-dose Antigen through Bronchial Expression of Macrophage-derived Chemokine. J. Exp. Med., 198: 533-543.
3.
Takeda, N., Arima, M., Tsuruoka, N., Okada, S., Hatano, H., Sakamoto, A., Kohno, K., and Tokuhisa, T. Bcl6 Is a Transcriptional Repressor for the IL-18 Gene.  J. Immunol., 171:426-431.
2.
Takenaga, M., Hatano, M., Yamashita, Y., Okada, S., Kuroda, Y., and Tokuhisa, T. Bcl6-dependent transcriptional repression by BAZF. Biochem. Biophys. Res. Commun., 303: 600-608.
1.
Obata, S., Okada, S., Ohkubo, Y., Murasawa, M., Hatano, M., Sekiya, S., and Tokuhisa, T. Overexpression of the c-fos gene perturbs functional maturation of M1 cells into macrophages.  Mol. Immunol., 39: 585-594.

2002
9.
Sasagawa, K., Matsudo , Y., Kang, M., Fujimura, L., Iitsuka, Y., Okada, S., Ochiai, T., Tokuhisa, T., and Hatano, M. Identification of Nd1, a novel murine kelch family protein, involved in stabilization of actin filaments.  J. Biol. Chem., 277: 44140-44146.
8.
Bahar, R., O-Wang, J., Kawamura, K., Seimiya, M., Wang, Y., Hatano, M., Okada, S., Tokuhisa, T., Watanabe, T., and Tagawa, M. Growth retardation, polyploidy and multinucleation induced by Clast3, a novel cell cycle-regulated protein.  J. Biol. Chem., 277: 40012-40019.
7.
Nakagawa, Y., Okada, O., Hatano, M., Ebara, M., Saisho, H., and Tokuhisa, T. Downregulation of bcl-xL is relevant to UV-induced apoptosis in fibroblasts.  J. Biochem. Mol. Biol. 35: 452-458.
*6.
Toyama , H., Okada, S., Hatano, M., Takahashi, Y., Takeda, N., Ichii, H., Takemori, T., Kuroda, Y., and Tokuhisa, T. Memory B cells without somatic hypermutation are generateded from Bcl6-deficient B cells.  Immunity, 17: 329-339.
5.
Arima, M., Toyama , H., Ichii, H., Kojima, S., Okada, S., Hatano, M., Cheng, G., Kubo, M., Fukuda, T., and Tokuhisa , T. A Putative Silencer Element in the IL-5 Gene Recognized by Bcl6.  J. Immunol., 169: 829-836.
*4.
Ichii, H., Sakamoto, A., Hatano, M., Okada, S., Toyama , H., Taki, S., Arima, M., Kuroda, Y., and Tokuhisa, T. Role for Bcl-6 in the generation and maintenance of memory CD8+ T cells. Nature Immunol., 3: 558-563.
*3.
Hikita, S., Hatano, M., Inoue, A., Sekita, N., Kobayashi, K., Ohtaki, M., Ogasawara, T., Okada, S., Hirasawa, H., and Tokuhisa, T. Overexpression of TIAP/m-survivin in thymocytes enhances cell proliferation.  Mol. Immunol., 39: 289-298.
2.
Seimiya, M., Bahar, R., Wang, Y., Kawamura, K., Tada, Y., Okada, S., Hatano, M., Tokuhisa, T., Saisho, H., Watanabe, T., Tagawa, M., and O-Wang, J. Clast5/Stra13 is a negative regulator of B lymphocyte activation.  Biochem. Biophys. Res. Commun., 292: 121-127.
1.
Murasawa, M., Okada, S., Obata, S., Hatano, M., Moriya, H., and Tokuhisa, T. GL7 defines the cycling stage of pre-B cells in murine bone marrow. Eur. J. Immunol., 32: 291-298.

2001
8.
Narita, M., Bahar, R., Hatano, M., Kang, M. M., Tokuhisa, T., Goto, S., Saisho, H., Sakiyama, S., and Tagawa, M. Tissue-specific expression of a suicide gene for selective killing of neurob lastoma cells using a promoter region of the NCX gene.  Cancer Gene Ther., 8: 997-1002.
7.
Yokohari, K., Yamashita, Y., Okada, S., Ohya, K., Oda, S., Hatano, M., Mano, H., Hirasawa, H., and Tokuhisa T. Isoform-dependent interaction of BRDG1 with Tec kinase. Biochem. Biophys. Res. Commun., 289: 414-420.
6.
Zhang, H., Okada, S., Hatano, M., Okabe, S., and Tokuhisa, T. A novel functional domain of Bcl6 family that recruits histone deacethylases. Biochim. Biophys. Acta, 1540: 188-200.
5.
Kang, M., Matsudo , Y., Sasagawa, K., Tokuhisa, T., and Hatano, H. Nd1, a novel murine Kelch family protein, may play a role as a housekeeping gene.  Biochim. Biophys. Acta, 1519: 167-174.
4.
Hartatik, T., Okada, S., Okabe, S., Arima, M., Hatano, H., and Tokuhisa, T. Binding of BAZF and Bcl6 to STAT6-binding DNA sequences. Biochem. Biophys. Res. Commun., 284: 26-32.
3.
Ogasawara, T., Hatano, M., Otaki, M., Kobayashi, K., Miyazaki , M., Nakajima, N., and Tokuhisa, T. A novel homologue of the TIAP/m-survivin gene.  Biochem. Biophys. Res. Commun., 282: 207-211.
*2.
Mizushima, N., Yamamoto, A., Hatano, M., Kobayashi, Y., Kabeya, Y., Suzuki, K., Tokuhisa, T., Ohsumi, Y., and Yoshimori, T. Dissection of autophagosome formation using Apg5-deficient mouse embryonic stem cells.  J. Cell Biol., 152: 657-668.
*1.
Kojima, S., Hatano, M., Okada, S., Fukuda, T., Toyama , Y., Yuasa, S., Ito, H., and Tokuhisa, T. Disruption of the Bcl6 gene increases testicular germ cell apoptosis in mice.  Development, 128: 57-65.

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