Despite significant Hes5 expression in thymus and thymus subsets, Hes5-deficient thymus contains normal cell numbers and normal proportions of T-cell subsets, but shows a higher percentage of B cells in the thymus. (A) RNA was prepared from whole thymus (T), and sorted thymus subsets DN (CD4⁻CD8⁻) and DN gated subsets DN1 (CD44⁺CD25⁻), DN2 (CD44⁺CD25⁺), DN3 (CD44⁻CD25⁺), and DN4 (CD44⁻CD25⁻). Hes1 and Hes5 mRNA levels were quantitated using RT-PCR. □ depicts mRNA levels in wild-type Hes5^+/+ thymus; ▩, mRNA levels in Hes5^−/− thymus. Values are means of 3 replicates plus or minus SEM. ND indicates not detected. (B) Mean (± SEM) total number of thymus cells from 13-Hes5 deficient mice and their littermates from 6 separate litters. (C) Surface expression of CD4 and CD8 antigens on thymus cells from a representative Hes5^−/− mouse and Hes5^+/+ littermate. Numbers in the corners represent the percentage of cells within the respective quadrant. (D,F) Top contour plots, showing surface staining for CD4 and CD8 antigens, depict the gated DN subset for representative Hes5^−/− and Hes5^+/+ littermates, while bottom contour plots depict CD19 and B220 antigen expression (D) and CD25 and CD44 antigen expression (F) by the gated DN population. (E,G) Bar graphs depict the mean percentage (± SEM) of gated DN cells expressing B220 and CD19 (E), CD44⁻CD25⁺ (DN3), and CD44⁻CD25⁻ (DN4) (G) for 11 Hes5^−/− mice and their Hes5^+/+ littermates. (H) Scatter plots depict the individual percentage (± SEM) and number of B220⁺CD19⁺ cells. (I) Scatter plots depict the mean percentage (± SEM) and number of B220⁺CD19⁺ cells in 3 Rag1^−/− recipients 4 weeks after transplantation of 2 × 10⁵ bone marrow cells from 6 Hes5^−/− and 6 Hes5^+/+ littermates.