B-lymphoid development is blocked in the transition from LY6D⁻ to LY6D⁺ CLP populations. (A) Representative FACS analysis displaying LY6D⁺ and LY6D⁻ populations within the Flt-3⁺IL-7R⁺Sca1^lowKit^low CLP from BM (femurs and tibias) of normal (IL-7^+/+) and IL-7–deficient (IL-7^−/−) mice. The numbers in the panels are mean percentages of LY6D^+/− cells of total CLP. (B) Collective data displaying absolute numbers of LY6D⁻ and LY6D⁺ CLP populations in wt (white bars) and IL-7–deficient mice (black bars). The data are mean + SEM, from 6 mice of each genotype, analyzed in 2 independent experiments. (C) Total cloning frequency (left) and lineage distribution (right) within BM CLPLY6D⁺ cells from IL-7^+/+ and IL-7^−/− mice. The CLPLY6D⁺ cells were sorted and manually plated at limiting doses (1, 2, and 5 cells/well for IL-7^+/+ and 2, 5, and 10 cells per well for IL-7^−/− cells) in 96-well plates. The frequencies were calculated based on numbers of positive wells using L-Calc software (StemCell Technologies, http://www.stemcell.com/tutorials/lcsetup.exe). The bars show mean 95% confidence interval. The difference between the 2 genotypes was compared by 2-tailed t test built in L-Calc software. For investigation of lineage potentials, the obtained cultures were analyzed by FACS for expressions of different lineage markers, including B cells (CD19⁺B220⁺), DCs (CD11C⁺), and NK cells (NK1.1⁺). The clones were defined as positive when having a minimum of 0.02% of each of the B, NK, and DC lineages and ≥ 10 gated events with an analysis of a minimum 25 000 blood cells. Bars are mean + SD. (D) Q-PCR analysis of expression of Ebf-1 in LY6D⁺ and LY6D⁻ CLPs from normal (IL-7^+/+) and IL-7–deficient (IL-7^−/−) mice. The data are normalized to the expression of Hprt, collected from 3 independent sorting experiments, and analyzed by triplicate Q-PCR reactions. The data are mean + SEM.