is responsible for stabilizing the basal precursor pool, enhancing their resistance to random perturbations. Further, it also accelerated the erythropoietic response to high Epo, an unexpect- ed effect for a negative regulator. Therefore, the dynamic properties of the Fas-mediated autoregulation offset the dynamic deficits of the slower, pO2/Epo-regulated feedback. Of interest, negative autoregulatory loops, in the context of simple transcrip- tional networks, were shown to accelerate the response to a stimulus, and to enhance network stability [32,33,34,35]. Our findings here suggest that these loops, which are abundant in biological systems, similarly improve the homeostasis and dynamic responses of lineage-specific progenitors in vivo. Materials and Methods Please also see Text S1: Supplementary Material and Methods section. Mice B6.MRL-Faslpr/J (Jackson Laboratories stock # 000482) were crossed with B6.129S7-Rag1tm1Mom/J (stock # 002216) to obtain homozygous double-mutant lpr-Rag12/2 mice (C57BL/6J back- ground). The homozygous double mutant gld-Rag12/2 mice (Balb/ C background) were obtained by crossing CPt.C3-Faslgld/J (Jackson Laboratories, stock # 002932) with C.129S7(B6)-Rag1tm1Mom/J (Jackson Laboratories stock # 003145). Other Balb/C mice for the hypoxia chamber experiments were purchased from Taconic. All experiments were approved by the University of Massachusetts Medical School IACUC committee, under protocol number A1586. Flow cytometry. Flow cytometry was performed as described [12] on freshly isolated spleen and bone-marrow cells. Dead cells were excluded using DAPI (Roche, Indianapolis, IN) or 7-AAD (BD Biosciences) viability dye. Data were analyzed using FlowJo software (Tree Star, Ashland, OR). Statistical analyses. Statistical analyses was performed using PASW (SPSS, Chicago, IL) and SAS (SAS, Cary, NC) statistical software. Data was log-transformed and analyzed by general linear mixed model ANOVA with gender and genotype as fixed effects and experiment as the random effect. Unadjusted p-values were reported in the figures for each gender-genotype group. Results Hypoxia and return to normoxia alter Fas expression and survival of early erythroblasts Erythropoietic stress results in suppression of Fas expression and reduced apoptosis in splenic ProE and EryA [12]. These findings suggested the model illustrated in Figure 1A, in which Fas- mediated cell death is a result of intercellular interactions between Fas and FasL-co-expressing cells within the spleen early erythro- blast compartment; these interactions are blocked by high Epo during stress (Fig 1B). Here we investigated this model further by housing mice in a hypoxic environment of 11% oxygen for 8 days, followed by a return to normoxia (21% oxygen). Hypoxia caused a sharp increase in plasma Epo (Fig 1C, lower left panel), suppressing Fas expression in EryA and enhancing their survival (Fig 1C, upper panel: example of flow-cytometric measurements of Fas expression and Annexin V binding; middle panels: summary of data form 2 to 9 mice per time point). Decreased apoptosis and Fas expression were associated with increased spleen EryA (Fig 1C, lower right panel). A return to 21% oxygen resulted in a rapid reversal, with Epo and EryA declining below their starting levels, and both Fas expression and apoptosis rising well above their starting basal Fas Stabilizes and Accelerates Erythropoiesis levels. These results suggest that modulation of Fas-mediated apoptosis by Epo plays a role in both the expansion of the EryA pool in response to hypoxia, and in its rapid contraction with the return to normoxia. Effect of reducing Fas-mediated apoptosis with Fas:Fc in vivo To examine the effect of Fas on EryA survival directly, we administered MyD882/2 mice with the purified chimeric molecule, Fas:Fc [36], which acts as a decoy receptor, binding FasL on the surface of EryA and blocking its ability to activate Fas. Control MyD882/2 mice were injected intraperitoneally with an equal volume of saline. The MyD882/2 strain was used in order to avoid potential reaction to contaminating bacterial Lipopoly- saccharide (LPS) in the Fas:Fc preparation [37]. A single administration of Fas:Fc (100 mg) resulted in a 20% reduction in unoccupied surface FasL in both splenic ProE and EryA, measured by binding of the Fas-blocking monoclonal antibody MFL3, directed against FasL [38] (not shown). This procedure decreased the number of Annexin V+ EryA from 70% in control to 50% in Fas:Fc -injected mice by 48 hours. There was an associated increase in EryA, and a doubling in blood reticulocytes (Fig S1). Reticulocytes, identified by their RNA content, mature within 24 hours [39]; a doubling of reticulocytes suggests a doubling of erythropoietic rate over the most recent 24 hours. These results support a causal relationship between Fas expression, EryA survival and erythropoietic rate. The dose/response characteristics of Epo-mediated Fas suppression and EryA expansion To assess the quantitative relationship between Epo, Fas suppression and EryA numbers we injected mice (n = 38) with varying doses of Epo, of between 1 and 300 Units /25 g body weight. We measured the Fas response on day 3, previously found to correspond to the lowest Fas expression level attained following acute Epo injection [12]. We found that EryA frequency in Ter119+ spleen cells was inversely related to the fraction of EryA cells that expressed Fas (Fig 2A, left panel). Half maximal suppression of Fas expression was seen in mice injected with 10 U/25 g (Fig 2A, right panel), corresponding to a doubling of EryA frequency. Complete suppression of Fas expression was seen at 30 U/ 25 g, and resulted in a 6-fold increase in EryA frequency. The frequency of EryA is inversely related to their Fas expression The relationship between EryA frequency, and the fraction of EryA that express Fas, may be fitted by a model in which EryA undergo Fas-mediated negative autoregulation (Fig 2B). We considered the frequency of EryA within the erythroblastic island, ‘A’, to be the result of three principal factors: first, a continuous input from earlier progenitors, ‘ß’; second, a continuous output, proportional to A,’aA’, into more differentiated progenitor subsets; and last, Fas-dependent cell death. We assumed that Fas-mediated cell loss would result when two EryA cells, expressing Fas and FasL respectively, interact within the erythroid compartment. The probability of such an encounter is proportional to the product of the frequencies of Fas+ and FasL+ EryA cells. This product is approximately equal to A2F, where ‘F’ denote the fraction of EryA cells that express Fas (Fig 2B; the fraction of EryA that express FasL in adult spleen is high enough that it can be approximated to 1; see Text S2: ‘Regulation of the EryA progenitor pool by Fas’). These considerations allow the steady-state level of EryA, at any given level of Fas, to be found by solving a quadratic equation. PLoS ONE | www.plosone.org 3 July 2011 | Volume 6 | Issue 7 | e21192