data by the fraction of Ter119+ cells per spleen and the spleen weight, for each individual mouse (Fig 5A). These data show significant increases, ranging from 1.5 to 4- fold, depending on genotype and sex, in both the frequency and absolute number of spleen CFU-e, ProE, EryA and EryB, in gld- Rag12/2 and lpr-Rag12/2 mice (Figures 4A–C and 5A). Specifically, all the splenic ProE, EryA and EryB subsets showed increased frequency within the Ter119high compartment in both gld-Rag12/2 and lpr-Rag12/2 mice relative to matched controls, with this increase reaching statistical significance in ten of the twelve comparisons made, namely ProE (p,0.02), EryA (p,0.002) and EryB (p,0.002) in female lpr-Rag12/2 mice, ProE (p,0.002), EryA (p,0.002) and EryB (p,0.02) in male lpr- Rag12/2 mice, ProE (p,0.02), EryA (p,0.02) and EryB (p,0.02) in female gld-Rag12/2 mice, and EryB (p,0.02) in male gld- Rag12/2 mice. The frequency of ProE and EryA in male gld- Rag12/2 mice was also increased relative to controls but did not reach statistical significance. Similarly, the absolute number of cells in each of these erythroid subsets increased in both gld- Rag12/2 and lpr-Rag12/2 mice, reaching significance in nine of the twelve comparisons made, namely, ProE (p,0.005), EryA (p,0.0005) and EryB (p,0.0005) in female lpr-Rag12/2 mice, ProE (p,0.0005), EryA (p,0.0005) and EryB (p,0.05) in male lpr-Rag12/2 mice, and ProE (p,0.05), EryA (p,0.005) and EryB (p,0.05) in female gld-Rag12/2 mice. The number of ProE, EryA and EryB in male gld-Rag12/2 mice also increased but the differences did not reach statistical significance (Fig 5A). The Fas Stabilizes and Accelerates Erythropoiesis number of gld-Rag12/2 mice available for analysis was smaller than that of the other sex/strain combinations (Fig 4B, 5C), possibly accounting for the failure of the change in their erythroblast subsets to reach statistical significance. Of note, there was no significant change in any of the equivalent subsets in bone-marrow (Fig 4D) for any sex/strain combination. These results are consistent with the pattern of erythroid Fas and FasL co-expression, which is largely restricted to spleen [12]. The largest increases were observed in ProE and EryA, in agreement with their higher Fas and FasL expression [12]. Variability in basal erythropoietic rate and in the size of spelnic erythroid subsets in gld-Rag12/2 and lpr-Rag12/2 mice Whilst the mean size of the ProE and EryA pools increased in gld-Rag12/2 and lpr-Rag12/2 mouse populations, their actual size in individual mice was highly variable. Using an F-test we found significantly higher variance in both the frequency and absolute number of nearly all erythroblast subsets in the gld-Rag12/2 and lpr-Rag12/2 mice, and in gld-Rag12/2 reticulocytes, compared with matched Rag12/2 controls (significantly different variance is marked with an ‘f’; Figures 3D, 4B–C, 5A). The EryA frequency distributions (Figures 5B and S2) show the larger spread of EryA pool size in the mutant mice. We also found increased coefficient of variation (CV) for EryA and ProE in male, but less so in female, gld-Rag12/2 and lpr-Rag12/2 mice (Figures 5B and S2). The coefficient of variation measures variability independently of the Figure 5. Loss of Fas function results in a larger and more variable basal spleen erythroid progenitor pool. (A) Spleen erythroid subsets ProE, EryA or EryB, expressed as absolute number of cells per gram body weight, in gld-Rag12/2, lpr-Rag12/2 and matched Rag12/2 controls, shown separately for male (M) and female (F) mice. Data corresponds to the same mouse dataset as in Fig 4. Box and whiskers delineate the central 50% and 90% of readings, respectively, with the median indicated with a horizontal line and arithmetic mean with a ‘+’. Data points correspond to individual mice. Between 11 and 40 mice examined per genotype; data pooled from several independent experiments. *p,0.05, **p,0.005, ***p,0.0005 (ANOVA, for difference in means); f = p,0.05, ff = p,0.005, fff = p,0.0005 (F test, for difference in variance). (B) Frequency distribution histograms for EryA, in male and female lpr-Rag12/2 and matched Rag12/2 controls. The coefficient of variation for each group is shown. Purple line is the fitted normal distribution curve. Same data set as in panel (A). doi:10.1371/journal.pone.0021192.g005 PLoS ONE | www.plosone.org 7 July 2011 | Volume 6 | Issue 7 | e21192