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Molecular and Cellular Biology, September 2009, p. 5128-5135, Vol. 29, No. 18
0270-7306/09/$08.00+0     doi:10.1128/MCB.00793-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

A Novel Gene Essential for the Development of Single Positive Thymocytes{triangledown}

Kiyokazu Kakugawa ,1,{dagger},{ddagger} Takuwa Yasuda,2,{dagger},{ddagger} Ikuo Miura,4,{ddagger} Ayako Kobayashi,2,{ddagger} Hitomi Fukiage,2,{ddagger} Rumi Satoh,1,{ddagger} Masashi Matsuda,3,{ddagger} Haruhiko Koseki,3,{ddagger} Shigeharu Wakana,4,{ddagger} Hiroshi Kawamoto,1*,{ddagger} and Hisahiro Yoshida2*,{ddagger}

Laboratory for Lymphocyte Development,1 Laboratory for Immunogenetics,2 Laboratory for Developmental Genetics, RIKEN Research Center for Allergy and Immunology, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa 230-0045, Japan,3 Technology and Development Team for Mouse Genotype Analysis, Japan Mouse Clinic RIKEN Bio-Resource Center, 3-1-1 Koyadai, Tsukuba-shi, Ibaraki 305-0074, Japan4

Received 18 June 2009/ Returned for modification 3 July 2009/ Accepted 8 July 2009

A critical step during intrathymic T-cell development is the transition of CD4+ CD8+ double-positive (DP) cells to the major histocompatibility complex class I (MHC-I)-restricted CD4– CD8+ and MHC-II-restricted CD4+ CD8– single-positive (SP) cell stage. Here, we identify a novel gene that is essential for this process. Through the T-cell phenotype-based screening of N-ethyl-N-nitrosourea (ENU)-induced mutant mice, we established a mouse line in which numbers of CD4 and CD8 SP thymocytes as well as peripheral CD4 and CD8 T cells were dramatically reduced. Using linkage analysis and DNA sequencing, we identified a missense point mutation in a gene, E430004N04Rik (also known as themis), that does not belong to any known gene family. This orphan gene is expressed specifically in DP and SP thymocytes and peripheral T cells, whereas in mutant thymocytes the levels of protein encoded by this gene were drastically reduced. We generated E430004N04Rik-deficient mice, and their phenotype was virtually identical to that of the ENU mutant mice, thereby confirming that this gene is essential for the development of SP thymocytes.

* Corresponding author. Mailing address for Hiroshi Kawamoto: Laboratory for Lymphocyte Development, RIKEN Research Center for Allergy and Immunology, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa 230-0045, Japan. Phone: 81-45-503-7010. Fax: 81-45-503-7009. E-mail: kawamoto{at}rcai.riken.jp. Mailing address for Hisahiro Yoshida: Laboratory for Immunogenetics, RIKEN Research Center for Allergy and Immunology, RIKEN Yokohama Institute, 1-7-22 Suehiro-cho, Tsurumi, Yokohama, Kanagawa 230-0045, Japan. Phone: 81-45-503-7059. Fax: 81-45-503-7058. E-mail: hisahiro{at}rcai.riken.jp

{triangledown} Published ahead of print on 20 July 2009.

{dagger} These authors contributed equally.

{ddagger} K. Kakugawa identified point mutations, made targeting vectors, and analyzed ENU mutant mice and GFP knock-in mice. T. Yasuda, H. Fukiage, A. Kobayashi, and H. Yoshida screened the ENU mutant pool by T-cell phenotype, mapped the responsible loci, and sequenced the candidate genes. I. Miura and S. Wakana established the ENU mutant founder library and used a high-throughput SNP method to regionalize the responsible loci. GFP knock-in mice were made by K. Kakugawa, M. Matsuda, and H. Koseki. Immunohistochemical analysis was done by R. Satoh. The paper was written by K. Kakugawa, H. Yoshida, and H. Kawamoto. All experiments were supervised by H. Yoshida and H. Kawamoto.

Molecular and Cellular Biology, September 2009, p. 5128-5135, Vol. 29, No. 18
0270-7306/09/$08.00+0     doi:10.1128/MCB.00793-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



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