the pre–B cell compartment in the mutant was significantly decreased compared with controls (Fig. 2 C). Indeed, the pre–B cell population in BMKO mice contained significantly more apoptotic (Annexin V+) cells than the control (Fig. 2 D). Recir- culating B220highIgM+ mature B cells were also reduced by three- fold in the mutant BM compared with controls, and apoptotic cells were increased by 1.5-fold (unpublished data).Thus, B cell devel- opment is partially blocked in the absence of Mule, and pre–B and mature B cells in BMKO BM are severely reduced as a result of increased apoptosis.When splenic B cells were examined, the percentage of B220+IgM+ B cells was reduced from 52.3% in controls to 38.5% in the mutant (Fig. 2 E, top). BMKO spleen contained 50% fewer cells than control spleen (Fig. 2 F, left). In addition, the B1a cell population that is normally present in the peritoneal cavity of control mice was virtually absent in BMKO mice (Fig. 2, E, [bottom] and F [right]).Thus, Mule is required for B cell development in the BM, as well as the homeostasis of ma- ture B cells in the spleen and B1a cells in the peritoneum. Impaired proliferation and activation of Mule-deficient B cells To determine whether impaired proliferation contributed to the reduced B cell numbers in BMKO mice, we performed 176 Figure 3. Impaired proliferation and activation of BMKO B cells. (A) Littermate control and BMKO mice were supplied with BrdU-containing drinking water for 3.5 d. Top left, total splenic B cells were stained with anti-BrdU and anti- B220 Abs and analyzed by flow cytometry (n = 5; **, P = 0.0021). Bottom left, gated B220+IgM+ cells were further gated on CD93+ and sorted to detect BrdU+ TS B cells (n = 4; *, P = 0.024). Top right, B220+IgM+ cells were gated on CD21+CD23low and sorted to detect BrdU+ MZ B cells (n = 4; ***, P = 0.000077). Bottom right, B220+IgM+ cells were gated on CD21+CD23+ and sorted for BrdU+ FO B cells (n = 4; ***, P = 0.0001). Numbers are the percentage of BrdU+ cells among the gated population. (B) Purified splenic littermate control and BMKO B cells were stimulated for 24 h with the indicated doses of anti-IgM (left) or anti-IgM plus anti-CD40 (right) and incubated with [3H]- thymidine. Results are the mean ± SD of triplicate. (C) Purified splenic littermate control and BMKO B cells were stimulated with 10 µg/ml anti-IgM or 10 µg/ml anti-IgM plus 0.5 µg/ml CD40 ligand for 24 h. Up-regulation of the indicated activation markers was monitored by flow cytometry. (D) Purified splenic littermate control and BMKO B cells were stimulated with 20 µg/ml anti-IgM for the indicated times. Lysates were immunoblotted to detect phospho-tyrosine (pTyr). (E) Splenic littermate control and BMKO B cells preloaded with Indo-1 were stimulated first with anti-IgM and then with thapsigargin (TG; positive control). The Ca2+ response was measured based on the ratio of violet (Ca2+ bound) to blue (Ca2+ free) fluorescence. Results (A–E) are representative of two to three independent experiments involving one to five mice per genotype. in vivo BrdU incorporation experiments and ob- served that the percentage of BrdU+ B cells among total splenic B cells of BMKO mice was only 50% of controls (Fig. 3 A, top left). To analyze BrdU in- corporation in various B cell subsets, B220+IgM+ cells from control and BMKO splenocytes were gated and then sorted by flow cytometry into CD93+ transitional (TS), CD21+CD23low marginal zone (MZ), and CD21+CD23+ follic- ular (FO) B cells. The majority of TS B cells were BrdU+ in both control and BMKO mice but the mutant showed signifi- cantly fewer of these cells (Fig. 3 A, bottom left). A more strik- ing deficit was found in the mutant MZ compartment, where the percentage of BrdU+ cells was reduced to 50% of that in controls (Fig. 3 A, top right). Mule-deficient FO B cells exhib- ited the most severe defect in proliferation, with the number of BrdU+ cells reaching only 30% of the control value (Fig. 3 A, bottom right). [3H]-thymidine incorporation assays confirmed that BMKO B cells proliferated much less vigorously than con- trol B cells in response to stimulation with anti-IgM or anti-IgM plus anti-CD40 (Fig. 3 B). Although the activation marker CD69 was up-regulated normally on stimulated BMKO B cells, levels of CD25 and CD86 up-regulation were decreased compared with controls (Fig. 3 C). Thus, Mule is required for splenic B cell proliferation and activation. The impaired B cell development in BMKO mice and defective proliferation of BMKO B cells in response to anti- IgM treatment suggested that Mule might be involved in BCR-induced signal transduction, which is initiated by protein E3 ligase Mule is essential for B cell homeostasis | Hao et al.