showing signs of illness were sacrificed the degree of splenomegaly was comparable (Figure S9A) and the histopathology of the lymphomas was similar. The majority of early-onset tumors, either from E2f2+/+ or E2f22/2 mice, featured high mitotic indices and extensive apoptosis with tingible body macrophages and a starry sky appearance similar to that of human Burkitt lymphoma (data not shown). In addition, B lineage cells (B220+) isolated from E2f2+/+ and E2f22/2 lymphomas exhibited comparable rates of proliferation and apoptosis (Figure S9B and S9C). Reduced populations of B lineage progenitors in E2f42/2 mice It has recently been shown that E2f4-deficient mice have defects that extend from early hematopoietic progenitor cells, through common lymphoid precursors and into the B and T lineages [40]. Specifically in the B lineage, E2f42/2 mice exhibit a partial block early in B lineage development prior to immunoglobulin gene rearrangement that results in a deficiency in the most mature pro- B subpopulation and a reduction in more mature B lineage cells [19] (Glozak et al., manuscript in preparation). Total viable white blood cell counts in the bone marrow were modestly higher for Em-myc E2f4+/+ and E2f4+/2 mice than for non- transgenic siblings. In the case of E2f42/2 mice, there was no increase associated with the Em-myc transgene and Em-myc E2f42/2 mice had about half as many cells as Em-myc wild type siblings (p = 0.0269). As expected, within the bone marrow, non-transgenic E2f42/2 mice had a lower proportion of B lineage cells compared to non-transgenic E2f4+/+ mice (P = 0.0133, Figure 6A). Strikingly, the usual expansion of the B lineage in response to the myc transgene failed to occur in the E2f42/2 mice. As a consequence, the proportion of B lineage cells in the bone marrow was significantly less in E2f42/2 Em-myc mice than in E2f4+/+ Em-myc mice (P = 0.0019). Myc did, however, elicit the usual reduction in the relative proportion of mature to less mature B lineage cells in the E2f4-deficient mice as in E2f4 wild type mice (Figure 6B). Motivated by the hypothesis that Em-myc lymphomas originate in early stage B lineage cells [34], we assessed progenitor populations in the E2f42/2 mice compared to their siblings. In CFU pre-B colony assays using bone marrow from non-transgenic E2F4 cohort mice, there were significantly fewer progenitors in E2f42/2 marrow than wild type marrow (data not shown; Glozak et al., manuscript in preparation). Notably, the pre-tumorous E2f42/2 Em-myc mice exhibited no expansion of immature B lineage cells (B220+ CD43+) as a proportion of the total bone marrow compared to their non- transgenic E2f42/2 siblings (Figure 6C). We suggest that the Myc transgene fails to overcome the inefficient developmental progression of the B lineage in E2f4-deficient mice, there is a reduced number of susceptible progenitor cells, and consequently delayed tumor emergence. As indicated by Figure 1D, E2f42/2 Em-myc mice displayed a much delayed tumor onset. Possibly because of this delay, of the twenty-six mice assessed, nine died before lymphoma emergence or were still healthy at analysis. The three mice that developed lymphoma early (within 150 days of birth) and one older mouse exhibited the standard Em-myc lymphoma phenotype, character- ized by an enlarged spleen and multiple enlarged lymph nodes. Thirteen mice developed lymphoma very late in life. Three of these mice exhibited lymphoma with modest spleen enlargement and isolated lymph node enlargement, similar the uncommon late onset lymphomas that occasionally develop in Em-myc mice wild type for E2F. The ten remaining E2f42/2 Em-myc mice displayed an atypical tumor phenotype that was only rarely noted in E2F wild type Em-myc mice (10 of 17 E2f42/2 mice compared to only 3 of 79 E2f4+/+ mice). These atypical tumors featured a loose tumor E2F and Myc-Induced Lymphoma Figure 6. E2f4 deficiency limits expansion of the B lineage in response to Myc. (A) The percentage of B lineage cells (B220+) within the bone marrow of normal and pretumorous E2F4 cohort mice with the indicated genotypes. (B) Proportions of bone marrow B lineage cell populations (B220+), from early to more mature stages, across the E2F4 cohort: surface immunoglobulin negative cells, IgM positive IgD negative B cells, IgM positive IgD positive B cells, and IgM negative IgD positive B cells. (C) The proportion of immature B lineage cells (B220+ CD43+) in the bone marrow of non-transgenic and pretumorous E2F4 mice of the indicated genotypes. doi:10.1371/journal.pgen.1000640.g006 PLoS Genetics | www.plosgenetics.org 9 September 2009 | Volume 5 | Issue 9 | e1000640