Primary, oncogenic K-Ras–expressing mice have a severe block in differentiation during early T-cell development. (A) Decreased thymic cellularity in oncogenic K-Ras–expressing mice (KM). Total thymocyte number of 4-week-old KM mice is reduced (P = .025) compared with control mice (8 days after pI-pC induction). (B) Histopathologic sections of the thymus of a primary mouse expressing oncogenic K-Ras (left) compared with normal control (right), 8 days after pI-pC induction (original magnification 40×; hematoxylin and eosin) show a perturbed thymic architecture with an increase in stromal tissue in the former. Shown is 1 representative example. (C) Expression of oncogenic K-Ras is associated with a decrease of the DP population and an increase of DN1 cells. Single-cell suspension from thymi of 4-week-old KM (n = 7) and control (n = 7) mice, 8 days after pI-pC induction, were prepared and stained for CD4 and CD8. CD25 and CD44 expression was analyzed gating on DN, DP, CD8 single-positive (SP), and CD4 SP populations. Compared with normal controls, DP cells were significantly reduced (P = .027, shown as percentage of live cells and P = .012 for total cell numbers), whereas DN1 and CD8 SP populations were significantly increased. Shown is 1 representative experiment of 7 independent experiments (top panel), and bar diagrams represent combined results (bottom: left, percentage of live cells; right, total cell number). DN indicates double-negative; DP, double-positive; and SP, single-positive. (D) DN cells of KM mice show reduced expression of CD3, TCR-β, and TCR-γ/δ. Histograms were gated on the DN population of thymocytes derived from KM or control mice. Shown is a representative example of 6 for each experiment. (E) Primary thymocytes exhibit enhanced apoptosis. DN-gated cells were analyzed with 7AAD and annexin V staining. Data are a representative of 3 independent experiments.