Expression of Gli3 is required for efficient transition from DN1 to DN2 in thymocyte development. (A) E13.5 thymocytes isolated from Gli3^+/+ and Gli3^–/– mice were gated on positive expression of the hematopoietic lineage-specific marker, CD45.2, to exclude cells of a nonhematopoietic origin from the analysis. (B) DN1 is overrepresented and DN2 underrepresented in E13.5 Gli3^–/– mice. CD45.2⁺ cells were analyzed for expression of CD44 and CD25. The percentages of DN1 to DN4 subsets are shown. Quadrants were defined according to DN subsets in E16.5 wild-type thymus. (C) Data from 8 litters showing the mean relative percentages of DN1 and DN2 cells in Gli3^–/–, Gli3^+/–, and Gli3^+/+ E13.5 embryos, expressed relative to the mean of the Gli3^+/+ littermates. Error bars represent standard error of the mean. (D) The c-kit expression was analyzed on E13.5 CD45.2⁺ thymocytes from Gli3^+/+ (left) and Gli3^–/– embryos (right). Percentages of CD45.2⁺c-Kit⁺ thymocytes are shown. (E) Thymocyte number isolated from E13.5 Gli3^+/+, Gli3^+/–, and Gli3^–/– embryos. There were no significant differences between genotypes.