Increased cell death in Hoxa-9^−/−fetal thymuses. (A) Microphotographs of sections of mutant (right) and normal (left) day-15.5 fetal thymuses stained with methylene blue (original magnification × 560). The arrow points to a pyknotic nucleus and the arrow heads mark areas of chromatin condensation. (B) Day-15.5 Hoxa-9^−/− thymocytes show a marked increase in hypodiploid nuclei by propidium iodide staining and FACS analysis. The percentage of hypodiploid nuclei in mutant thymuses is dramatically increased (lower two panels) as compared with control adult and fetal thymuses (upper two panels). (C) Annexin V staining of fetal thymocytes. Mutant thymuses (n = 7) show an approximate threefold increase in the percentage of cells binding high levels of annexin V as compared with control animals (n = 10), as well as significant increases in the fraction binding intermediate levels. (D) Bcl-2 protein levels in day-15.5 thymocytes. Control thymocytes (n = 12) show high levels in bcl-2 protein in nearly all cells, whereas half of the Hoxa-9^−/− thymocytes (n = 4) show markedly reduced levels of bcl-2 (solid lines). Dotted lines depict staining with hamster IgG-PE as a negative control.