Increased surface pre-TCR levels, apoptosis, and premature differentiation in dnNb TG thymocytes. (A) Hes-1 gene expression levels, measured by RT-PCR, in WT (black) and dnNb TG (gray) thymocytes (top), or WT (black) and Numb TG (gray) T cells (bottom). (B) Pre-TCR (top), CD5 (middle), and Annexin V (bottom) flow cytometric measurement of DN3, DN4, or total DN WT or dnNb TG thymocytes; WT (solid line) and dnNb TG (broken line). (C) Pre-TCR (top), CD5 (middle), and CD25 (bottom) flow cytometric measurement in WT or dnNb TG DP thymocytes; WT (solid line) and dnNb TG (broken line). (D) Confocal images of fetal thymic frozen sections treated with TOPRO-3 to detect DNA. Apoptotic nuclei are marked with red arrows. (E) High magnification images of the nuclei marked with red arrows in panel D. (F) Average number of metaphases (left) and telophases (right) per fetal thymic section of WT (black), dnNb TG (dark gray), or Numb TG (light gray) mice. All data are representative of at least 3 independent experiments.