population mean. Overall, CV values for all splenic erythroblast subsets and for peripheral blood reticulocytes in both gld-Rag12/2 and lpr-Rag12/2 male and female mice, are significantly increased (p = 0.017, Figure S2 B). These findings indicate that a key function of Fas-mediated negative autoregulation is to suppress variability in the steady-state precursor pool and in erythropoietic rate, thus stabilizing the basal state. A delayed response to Epo-driven erythropoietic stress in gld-Rag12/2 and lpr-Rag12/2 mice We took two approaches to assess the stress response of the gld- Rag12/2 and lpr-Rag12/2 mice. First, we injected mice with a single high dose of Epo (300 U/ 25 g body weight), and followed the resulting increase in erythropoietic rate for 6 days (Fig 6A–D). Initially, on days 1 and/or 2, hematocrit was higher in both gld- Rag12/2 and lpr-Rag12/2 mice compared with controls, by 1– 2%, possibly reflecting the larger basal erythroblast pool in these mice. However, between days 2 and 3, there was a significantly faster increase in hematocrit in control mice, which rose by over 4% in the space of 24 hours, overtaking the hematocrit of gld- Rag12/2 and lpr-Rag12/2 mice (Fig 6A, 6D). There was no equivalent rapid increase in hematocrit in the gld-Rag12/2 and lpr-Rag12/2 mice. By subtracting the mean hematocrit on day 2 from the mean hematocrit on day 3, in 5 independent comparisons between independent experiments on those days, we found that the rate of change in the hematocrit was significantly higher in control mice (p,0.005, Fig 6D). The slower increase in hematocrit in both gld-Rag12/2 and lpr-Rag12/2 mice at this time was due to a slower expansion of EryA precursors, in spite of their larger basal EryA pools (Fig 6B, 6C). The difference in EryA expansion was largest on days 2 (lpr-Rag12/2 mice) and 3 (gld-Rag12/2 mice) (Fig 6C), corresponding to the time when Epo- mediated Fas suppression in wild-type mice reaches its peak [12]. Of note, EryA expansion in control mice was a massive, 30 to 60 fold increase over the basal EryA pool. The shortfall in EryA on day 2 in the lpr-Rag12/2 mice was equivalent to 10 times the size of the basal EryA pool, or 30% of the total expansion in control mice on that day (Figure 6B,C, p,0.05; the size of the basal EryA pool is marked Fig 6C as a black bar). A similar delay in EryA expansion was seen in gld-Rag12/2 mice (Figure 6B,C). Further, injection of a much lower Epo dose (10 U/ 25 g body weight) in lpr-Rag12/2 mice again resulted in delayed hematocrit and ProE responses (Fig S3). Therefore, the presence of Fas-mediated negative autoregulation accelerates the erythropoietic response over a wide Epo stress range. Analysis of changes in the ProE population showed similar results. In spite of its larger size in the basal state, expansion of the ProE pool in both gld-Rag12/2 and lpr-Rag12/2 mice was slower between days 1 and 3, the differences in the absolute size of the pools reaching significance on days 1 (gld-Rag12/2 mice) and 3 (lpr-Rag12/2 mice) (Fig 7A). Furthermore, the rate of increase in ProE in response to Epo peaked in all mice between days 1 and 2, attaining a significantly lower level in gld-Rag12/2 and lpr-Rag12/ 2 mice compared with matched controls (p = 0.00004, Fig 7 B,C). A delayed erythropoietic stress response to reduced atmospheric oxygen in gld-Rag12/2 mice In a second approach, we examined the response of the gld- Rag12/2 mice to an acute reduction of atmospheric oxygen to 11% (Fig 8). The response to reduced atmospheric oxygen is complex since, in addition to elevating serum Epo, it stimulates additional cytokines as well as changes in ventilation and plasma volume that may indirectly alter erythropoietic responses Fas Stabilizes and Accelerates Erythropoiesis [8,44,45]. Furthermore, the actual increase in serum Epo in response to lower atmospheric oxygen is determined not only by the initial hypoxic stimulus, but also by the ensuing erythropoietic response, which determines the duration of tissue hypoxia (Fig 1A). We chose to examine gld-Rag12/2 mice, since, unlike the lpr- Rag12/2 strain, their starting baseline Epo levels are very similar to those of their matched controls (Fig 3B). We found that in spite of their higher starting hematocrit and reticulocyte count, the increase in erythropoietic rate in gld-Rag12/2 mice was significantly slower, as seen by a significantly delayed increase in hematocrit (Fig 8A) and a lower reticulocyte count (Fig 8B). There was a correspondingly slower increase in EryC erythroblasts (Fig 8D), at both 24 and 72 hours. This sluggish response presumably prolonged the tissue hypoxia in these mice, as reflected by their higher serum Epo at 72 hours (Fig 8C). These results clearly show a delayed stress response in mice lacking Fas- mediated negative autoregulation of erythroblasts. Unlike the response to injection of a fixed, high Epo dose (Fig 6), the slower erythropoietic response to hypoxia in the gld-Rag12/2 mice was not associated with a difference in the size of the ProE/ EryA pools at the early (24 hour) time point. Such a difference may have occurred earlier; alternatively, hypoxia may accelerate the maturation of EryA cells, so that reserve, Fas+ EryA cells in control mice do not contribute to an increase in the EryA pool but instead differentiate rapidly and contribute to the increase that we see in the EryC pool by 24 hours (Fig 8D). Indeed, it has been observed previously that hypoxic stress as a result of bleeding or phehylhydrazine stress accelerates erythroblast maturation [46,47,48]. The larger number of EryA in gld-Rag12/2 by 72 hours presumably reflects the higher Epo level at that time, in turn a result of the more prolonged stress these mice presumably experience, due to their sluggish erythropoietic response to the initial hypoxic stimulus. Discussion We investigated the role of Fas and FasL-mediated negative autoregulation in the early erythroblast pool. We generated gld- Rag12/2 and lpr-Rag12/2 mouse strains, deficient in FasL and Fas, respectively, bred onto two distinct genetic backgrounds. Both these strains showed similar erythropoietic deficits. They confirm the hypothesis that Fas and FasL are negative regulators of splenic EryA and ProE, resulting in negative regulation of erythropoiesis at the whole animal level. In addition, they show a striking, non- redundant role for Fas in stabilizing basal erythropoiesis. Surprisingly, in spite of the removal of a negative regulator and the consequent larger basal precursor pool, the response of the mutant mice to erythropoietic stress was significantly delayed. These findings reveal that an autoregulatory loop local to erythropoietic tissue can exert key dynamic properties on erythropoiesis as a whole. They also provide experimental evidence that regulation of erythroid precursors through apoptosis, though apparently wasteful, accelerates the response to erythro- poietic stress. The presence of a local negative autoregulatory loop in spleen erythropoietic tissue Several lines of evidence suggest that EryA and ProE negatively regulate their own survival through their Fas and FasL-mediated interactions. First, the pattern of co-expression of both Fas and FasL by EryA and ProE [12,31]; second, the close apposition of cells within the erythroblastic island, making an interaction between Fas+ and FasL+ cells possible [31]; and last, our finding that a mathematical model in which EryA negatively regulate their PLoS ONE | www.plosone.org 8 July 2011 | Volume 6 | Issue 7 | e21192