Fas Stabilizes and Accelerates Erythropoiesis and the erythroid progenitor and precursor pools. We first examined the hematocrit (the fraction of all blood volume that is attributable to red blood cells), blood reticulocytes and plasma Epo. lpr-Rag12/2 mice on the C57BL/6 background had a normal reticulocyte count and normal hematocrit (Figure 3A, top left panel, Table S1). However, plasma Epo was significantly lower, by 35%, than in control mice (p = 0.001; Epo = 9.060.8 mU/ml, mean 6sem, in lpr-Rag12/2, and 13.761.0 for Rag12/2 controls; Fig 3A, bottom left panel, and Fig 3B). Therefore, lpr-Rag12/2 mice compensate for the chronic absence of a negative regulator through the pO2/Epo negative feedback loop, decreasing Epo concentration so as to avoid an unnecessary increase in hematocrit. By contrast, the gld-Rag12/2 mice, on the Balb/C background, had a significantly elevated hematocrit (p,0.00001; hematocrit = 52.060.3% vs. 49.860.4% for gld-Rag12/2 vs. Rag12/2 respec- tively, mean6sem, Figures 3A; a similar difference was found in a second group of mice assayed using a Coulter counter, Table S1). Reticulocyte count was also more than double that of con- trols (p,0.0001; Reticulocyte count = 1.4060.14% vs. 0.596 0.04% for gld-Rag12/2 vs. Rag12/2 respectively, Fig 3C–D), and hemoglobin concentration was also significantly elevated in 2 independent experiments (Table S1). As comparison, mice we housed for 3 weeks in 12% oxygen, equivalent to an altitude of 14,000 feet, increased their hematocrit from 51.660.2 to 57.860.5%, an increase of 6%. Therefore, the 2.2% increase in Figure 3. Increased erythropoiesis in mice deficient in the Fas pathway. Legend in A also applies to panels B, D. lpr-Rag12/2 mice are on the C57BL/6 background (in blue), and are compared with control Rag12/2 mice on the C57BL/6 background. gld-Rag12/2 mice are on the Balb/C background (in red), and are compared with control Rag12/2 mice on the Balb/C background. (A) Hematocrit ( = fraction of the blood volume that is due to red cells) and Plasma Epo of lpr-Rag12/2, gld-Rag12/2 and Rag12/2 age and strain-matched control mice. M = males. F = females. Box and whiskers delineate the central 50% and 90% of readings, respectively. Median is indicated with a horizontal line; arithmetic mean with a ‘+’. Data points correspond to individual mice. Between 11 and 40 mice examined per genotype. *p,0.05, **p,.005, ***p,0005 (ANOVA). (B) Hematocrit vs. plasma Epo in the subset of mice where both values were measured, in the basal state, for lpr-Rag12/2 and matched Rag12/2 control mice (left panel), and for gld-Rag12/2 and matched Rag12/2 controls (right panel). Data are mean 6 sem of $16 mice *p#0.001 (two-tailed t test, unequal variance). (C) Flow cytometric measurement of reticulocyte number. Top: whole blood stained with either DRAQ5 (detects DNA) or thiazole orange (TO, detects both DNA and RNA). Reticulocytes lack a nucleus but retain RNA. They therefore form a DRAQ5-negative, TO-positive population. Bottom panel shows analysis in wild-type (WT) mice either in the basal state or following Epo injection; and in gld-Rag12/2 and control Rag12/2 mice. (D) Reticulocyte in lpr-Rag12/2, gld-Rag12/2 and matched Rag12/2 controls, measured by flow-cytometry ***p,0.0001, two-tailed t-test, unequal variance; ff = p,0.001, F test. doi:10.1371/journal.pone.0021192.g003 PLoS ONE | www.plosone.org 5 July 2011 | Volume 6 | Issue 7 | e21192