Inhibition of Rho at different stages of thymocyte development gives different perspectives on Rho function

Abstract

Development of thymocytes can be staged according to the levels of expression of the cell-surface markers CD4, CD8, CD44, CD25 and CD2. Thymocyte development is regulated by a complex signalling network [1], one component of which is the GTPase Rho. The bacterial enzyme C3 transferase from Clostridium botulinum selectively ADP-ribosylates Rho in its effector-binding domain and thereby abolishes its biological function [2,3]. To explore the function of Rho in thymocyte development, we previously used the proximal promoter of the gene encoding the Src-family kinase p56lck to make transgenic mice that selectively express C3 transferase in the thymus [4–6]. In these mice, which lack Rho function from the earliest thymocyte stages, thymocyte numbers are reduced by approximately 50- to 100-fold. Here, we describe transgenic mice that express C3 transferase under the control of the locus control region (LCR) of the CD2 gene; this regulatory element drives expression at a later stage of thymocyte development than the lck proximal promoter [7]. In these mice, thymocyte numbers were also reduced by 50- to 100-fold, but unlike the lck–C3 mice, in which the reduction predominantly results from defects in cell survival of CD25+ thymocyte progenitors, the CD2–C3 transgenic mice had a pre-T-cell differentiation block at the CD25+ stage after rearrangement of the T-cell receptor (TCR) β chains. Analysis of CD2–C3 mice demonstrated that Rho acts as an intracellular switch for TCR β selection, the critical thymic-differentiation checkpoint. These results show that Rho-mediated survival signals for CD25+ pre-T cells are generated by the extracellular signals that act on earlier thymocyte precursors and also that temporal cell-type-specific elimination of Rho can reveal different functions of this GTPase in vivo.