Functional Runx1 deletion by targeted excision by Tg(vav-Cre) or Cd79a^hCre/+ expression leads to profound defects in B-cell development. (A) Schematic representation of B-cell development starting from the HSC compartment. B-cell specification occurs at the CLP-B stage (Ly6D⁺), before the expression of the classical B-cell antigens CD19 and B220. The stage in B-cell development at which Tg(vav-Cre) and Cd79a^hCre/+ mediate excision is indicated (see also supplemental Figure 2). Receptors necessary for the proliferative expansion of the indicated cell types are depicted. (B) FACS analysis of lineage-depleted BM cells to detect HSC (Lin^negSca1⁺Kit^hi), myeloid multipotent progenitor (Lin⁻Sca1⁻Kit^hi), and lymphoid-primed multipotent progenitors (LMPP) (Lin^negScal⁺Kit^hiFlt3^hi) compartments in vav-Cre mediated excision. The total cell number determined for each analyzed mouse (2 femora) is depicted by a dot, and the median for the indicated number of mice is shown. (C) FACS strategy for detection of total CLP (Lin^negIl7r⁺Flt3^hiKit^medSca1^med) and the percentage therein specified for B-cell development (Ly6D⁺) from lineage-depleted BM cells. Each dot represents the total number of CLPs per Tg(vav-Cre) mouse (2 femora). The percentage of Ly6D⁺ cells within the CLP fraction of either (upper graph) Tg(vav-Cre) or (lower graph) Cd79a^hCre/+ mice is indicated. Representative FACS analysis of total CLPs analyzed for Ly6d expression is shown on the right. (D) FACS analysis of CD19⁺Lin2^neg cells of the BM, detecting pro-B (B220^medCD43^hiIgL^neg), pre-B cells (B220^medCD43^med/loIgL^neg), immature B cells (B220^medCD43^med/loIgL⁺), and mature (recirculating) B cells (B220^hiCD43^negIgL⁺) of Cd79a^hCre/+ mice. Graph depicts the percentage of pro-B/pre-B cells within the total BM of mice with the indicated genotypes. P values were calculated by a nonpaired Student t test.