A

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 / (TCR/) ex-pressed on their surface (3, 5, 16, 17). By using reverse geneti