A Novel Gene Essential for the Development of Single Positive Thymocytes

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.The differentiation step from the double-positive (DP) to single-positive (SP) thymocyte stage is critically regulated by signals originating from the T-cell receptor α/β (TCRα/β) expressed on their surface (3, 5, 16, 17). By using reverse genetic approaches by knocking out or overexpressing various genes that are expected to be involved in TCR signaling, including its ligand major histocompatibility complex molecules and coreceptors CD4 and CD8, the roles of these genes in T-cell development have been investigated intensively (11, 12). However, to identify totally unknown mechanisms in T-cell development, the forward genetic approach is required. N-ethyl-N-nitrosourea (ENU) is a potent mutagen that randomly induces point mutations throughout the genome in a dose-dependent manner, and ENU mutagenesis has been a representative forward genetic strategy (4, 15). We have been screening phenotypes of ENU-mutagenized mice, focusing on defects in T-cell development.