could ameliorate the absence of Fas or FasL include upregulation of alternative negative regulators such as ERK1 [49], attenuation of alternative EpoR anti-apoptotic pathways such as Stat5-induced bcl-xL, phosphoinositol-3 kinase/AKT and suppression of Bim and Foxo3a [17,24,51], or a decrease in factors that stimulate erythropoiesis such as BMP [52]. A stabilizing function for the Fas and FasL-mediated negative autoregulatory loop Random variation in the number of progenitors is an inevitable consequence of inherent fluctuations in biological systems [53,54,55], found in mice that are genetically identical, of similar age and housed in similar stable conditions. The pO2/Epo- mediated negative feedback loop (Fig 1A) adjusts overall mean erythropoietic rate in the face of such fluctuations over time, but the intrinsic delay in this loop may result in oscillations and limit the system’s stability. Here we found that in the absence of Fas or FasL, there was a significant increase in variability, reflected by both increased variance and increased coefficient of variation, of splenic ProE, EryA and EryB in individual mice, compared with control mice (Fig 4B, Fig 5A). Bone-marrow erythroid subsets were not affected, but there was increased variability of overall erythropoietic rate, as reflected by the reticulocyte counts. Therefore, Fas-mediated autoregulation in spleen has a stabilizing effect on erythropoiesis at the whole animal level, offsetting the limitations of the pO2/Epo-mediated negative feedback loop. The stabilizing influence of the Fas/FasL interaction is a result of its sensitivity to the size of the erythroblast pool. For comparison, a recently identified cell-autonomous negative regulator of splenic erythropoiesis in vivo, ERK1, does not appear to contribute to the stability of erythropoiesis [49]. Fas -mediated apoptosis of ProE or EryA depends on the probability that two cells expressing Fas and FasL respectively, encounter each other within the erythropoietic niche. This probability is dependent on their frequency in tissue (Fig 2 and Text S2). Should their frequency be in excess, Fas-mediated loss of EryA would accelerate, providing an automatic correction. Conversely, a shortage of EryA would lower the probability of their interaction and death, allowing their number to increase. Both negative and positive corrections take place locally in erythropoietic tissue, with little delay and without the need to engage the pO2/Epo-mediated negative feedback loop, avoiding potentially deleterious corrective swings in systemic tissue pO2. Fas-mediated Negative autoregulation accelerates the stress response Unexpectedly for mice lacking a negative erythropoietic regulator, the response of gld-Rag12/2 and lpr-Rag12/2 mice to acute Epo administration was delayed. The splenic EryA pool expanded 30 fold its basal size in control mice by day 2; this massive increase was reduced by 30% in the gld-Rag12/2 and lpr- Rag12/2 mice, reflecting a significant shortfall, equivalent to 10 times the size of the basal EryA pool. This shortfall occurred in spite of a larger than normal basal EryA pool in the mutant mice. We found a similar delay in the expansion of ProE cells in the gld- Rag12/2 and lpr-Rag12/2 mice. To explain this phenomenon, we propose the model illustrated in Figure 9. EryA are continuously formed from earlier progenitors ( = EryA input) but the majority dies, with only a small fraction remaining as the basal EryA pool. However, the apoptosis-prone EryA form a reserve population that may be rapidly recruited by high Epo during stress. We propose that the rapid, 30-fold expansion in EryA within 2 days of Epo Fas Stabilizes and Accelerates Erythropoiesis administration corresponds to the size of the reserve EryA population. The smaller initial expansion in the gld-Rag12/2 and lpr-Rag12/2 mice suggests that part of the EryA reserve is regulated through Fas, and is missing in these mutant mice. These observations suggest that the high apoptotic rates of ProE and EryA in the basal state, in part due to Fas, provide a mechanism that accelerates the stress response. The rescue of ProE/EryA from apoptosis allows a faster increase in their number compared with their generation by cell division from earlier progenitors. This faster increase accelerates erythropoietic rate and prevents a prolonged tissue hypoxia. We found that the response of the erythropoietic system to reduced atmospheric oxygen was similarly delayed in gld-Rag12/2 mice, as reflected by lower reticulocyte counts and a slower increase in hematocrit. In this more complex, hypoxic stimulus to the system, we noted that the principal difference in the erythroblast pools between gld- Rag12/2 and control mice appeared at the EryC stage, rather than in the earlier, ProE and EryA stages. We suggest that this may be due to accelerated maturation of erythroblasts, noted in the older literature in response to hypoxic stress [46,47,48]. Of note, in ERK12/2 mice, the stress response is timely and somewhat amplified [49]. Unlike Fas-mediated autoregulation, which is exerted at the level of erythroblasts, ERK1 appears to suppress a much earlier, BMP-dependent BFU-e progenitor, which is therefore unlikely to contribute to the initial, acute phase of stress. Figure 9. Absence of a Fas-regulated EryA reserve delays the response to stress. EryA cells are continuously formed from earlier precursors (‘input’). In the basal state, when Epo concentrations are low, only a small fraction of these cells survive, forming the ‘basal EryA pool’ (in purple). The remaining EryA undergo apoptosis, either through Fas (‘Fas-regulated reserve’, green) or alternative mechanisms (‘Alternative reserve’, blue). Together, the EryA reserve pools are 30 to 60 fold the size of the basal pool (see Figure 6). During the initial response to stress, high Epo levels rescue the EryA reserve pools from apoptosis, resulting in an immediate increase in the size of the surviving EryA pool and an increase in erythropoietic rate (solid colors indicate surviving cells, dashed lines indicate cells that underwent apoptosis). We suggest that lpr and gld mice partially compensate for the absence of the Fas- regulated reserve by generating fewer EryA cells (a smaller input). In this way, the absence of Fas –mediated apoptosis does not excessively increase the basal EryA pool (which does increase 1.5–4 fold, see Figure 5; this increase is much smaller than the stress-induced increased and is not shown). During stress, the absence of the Fas-regulated reserve in lpr and gld mice reduces the number of EryA that may be immediately recruited into the surviving EryA pool and consequently delays the stress response. doi:10.1371/journal.pone.0021192.g009 PLoS ONE | www.plosone.org 12 July 2011 | Volume 6 | Issue 7 | e21192