Summary BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, while expression of the erythroid regulator GATA1 directs SMAD1 loss on non-erythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.

In thalassemia and other iron loading anemias, ineffective erythropoiesis and erythroid signaling molecules are thought to cause inappropriate suppression of a small peptide produced by hepatocytes named hepcidin. Previously, it was reported that the erythrokine GDF15 is expressed at very high levels in thalassemia and suppresses hepcidin expression. In this study, erythroblast expression of a second molecule named twisted gastrulation (TWSG1) was explored as a potential erythroid regulator of hepcidin. Transcriptome analyses suggest TWSG1 is produced during the earlier stages of erythropoiesis. Hepcidin suppression assays demonstrated inhibition by TWSG1 as measured by quantitative polymerase chain reaction (PCR) in dosed assays (1-1000 ng/mL TWSG1). In human cells, TWSG1 suppressed hepcidin indirectly by inhibiting the signaling effects and associated hepcidin up-regulation by bone morphogenic proteins 2 and 4 (BMP2/BMP4). In murine hepatocytes, hepcidin expression was inhibited by murine Twsg1 in the absence of additional BMP. In vivo studies of Twsg1 expression were performed in healthy and thalassemic mice. Twsg1 expression was significantly increased in the spleen, bone marrow, and liver of the thalassemic animals. These data demonstrate that twisted gastrulation protein interferes with BMPmediated hepcidin expression and may act with GDF15 to dysregulate iron homeostasis in thalassemia syndromes. IntroductionSystemic iron homeostasis in mammals is largely maintained by the effects of hepcidin, 1 a small protein produced by hepatocytes. Hepcidin is regulated at the transcriptional and posttranscriptional levels by multiple extracellular signals related to iron homeostasis and inflammation. Erythropoiesis is also thought to regulate hepcidin expression through a variety of mechanisms including anemia-related hypoxia and erythropoietin production. ␤-Thalassemia syndromes are congenital anemias caused by mutations that reduce or abolish ␤-globin gene expression. Despite the common feature of decreased globin chain synthesis in all patients, there are prominent phenotypic variations in the disease that are not fully understood. 2 In so-called "iron-loading" anemias like thalassemia, the diseased erythron dysregulates iron homeostasis by inhibiting hepcidin expression even in the presence of severe iron overload. Humans with thalassemia syndromes express very high levels of a cytokine named GDF15, and GDF15 present in thalassemia patients' sera inhibited hepatic hepcidin expression ex vivo. 3 However, thalassemia sera also suppressed hepcidin expression to a lesser degree after immunoprecipitation of GDF15. 3 It was therefore hypothesized that GDF15 may act with other molecules to suppress hepcidin.In addition to clinical research in humans, murine models were developed for studies of thalassemia and hepcidin regulation. Mice with deletions of both the ␤ minor and ␤ major genes (th3 genotype) have a ␤-thalassemia intermedia phenotype in the heterozygous state. The homozygous deletion (th3/th3) results in death...

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Purpose of review Acute anemic stress induces a physiological response that includes the rapid development of new erythrocytes. This process is referred to as stress erythropoiesis, which is distinct from steady state erythropoiesis. Much of what we know about stress erythropoiesis comes from the analysis of murine models. In this review, we will discuss our current understanding of the mechanisms that regulate stress erythropoiesis in mice and discuss outstanding questions in the field. Recent findings Stress erythropoiesis occurs in the murine spleen, fetal liver and adult liver. The signals that regulate this process are Hedgehog, bone morphogenetic protein 4 (BMP4), stem cell factor and hypoxia. Recent findings show that stress erythropoiesis utilizes a population of erythroid-restricted self-renewing stress progenitors. Although the BMP4-dependent stress erythropoiesis pathway was first characterized during the recovery from acute anemia, analysis of a mouse model of chronic anemia demonstrated that activation of the BMP4-dependent stress erythropoiesis pathway provides compensatory erythropoiesis in response to chronic anemia as well. Summary The BMP4-dependent stress erythropoiesis pathway plays a key role in the recovery from acute anemia and new data show that this pathway compensates for ineffective steady state erythropoiesis in a murine model of chronic anemia. The identification of a self-renewing population of stress erythroid progenitors in mice suggests that therapeutic manipulation of this pathway may be useful for the treatment of human anemia. However, the development of new therapies will await the characterization of an analogous pathway in humans.

The Friend virus susceptibility 2 (Fv2) locus encodes a dominant host factor that confers susceptibility to Friend virus-induced erythroleukaemia in mice. We mapped Fv2 to a 1.0-Mb interval that also contained the gene (Ron) encoding the stem cell kinase receptor (Stk). A truncated form of Stk (Sf-stk), which was the most abundant form of Stk in Fv2-sensitive (Fv2ss) erythroid cells, was not expressed in Fv2 resistant (Fv2rr) cells. Enforced expression of Sf-stk conferred susceptibility to Friend disease, whereas targeted disruption of Ron caused resistance. We conclude that the Fv2 locus encodes Ron, and that a naturally expressed, truncated form of Stk confers susceptibility to Friend virus-induced erythroleukaemia.

The erythroid response to acute anemia relies on the rapid expansion in the spleen of a specialized population of erythroid progenitors termed stress BFU-E. This expansion requires BMP4/Madh5-dependent signaling in vivo; however, in vitro, BMP4 alone cannot recapitulate the expansion of stress BFU-E observed in vivo, which suggests that other signals are required. In this report we show that mutation of the Kit receptor results in a severe defect in the expansion of stress BFU-E, indicating a role for the Kit/SCF signaling pathway in stress erythropoiesis. In vitro analysis showed that BMP4 and SCF are necessary for the expansion of stress BFU-E, but only when spleen cells were cultured in BMP4 ؉ SCF at low-oxygen concentrations did we recapitulate the expansion of stress BFU-E observed in vivo. Culturing spleen cells in BMP4, SCF under hypoxic conditions resulted in the preferential expansion of erythroid progenitors characterized by the expression of Kit, CD71, and TER119. This expression pattern is also seen in stress erythroid progenitors isolated from patients with sickle cell anemia and patients with ␤-thalassemia. Taken IntroductionAcute anemia induces a systemic response that involves the induction of erythropoietin (Epo) expression in the kidney and the rapid mobilization and differentiation of erythroid progenitors. Previous work from our laboratory led to the development of a new model for the recovery from acute anemia where BMP4 expression in the spleen drives the expansion of a specialized population of stress erythroid progenitors, which we termed stress BFU-E. 1 Our analysis of flexed-tail (f) mutant mice, which exhibit a defect in BMP4-dependent signaling, demonstrated that BMP4 is required for the recovery from acute anemia. 1 BMP4 is required only transiently and acts on an earlier progenitor, the BMP4 responsive (BMP4 R ) cell, causing it to differentiate into a stress BFU-E. BMP4 R cells are contained in the spleen megakaryocyte erythroid progenitor (MEP) population but are not observed in bone marrow MEPs. We define a stress BFU-E as a progenitor that rapidly forms a large burst colony in 5 days when cultured in media containing only Epo. If stress BFU-Es are cultured for 7 days as steady state bone marrow BFU-Es are, they produce significantly larger colonies. 1 We showed that treatment of spleen cells with BMP4 in vitro resulted in the expansion of stress BFU-Es, which demonstrated that BMP4 R cells are resident in the spleens of normal mice. However, culturing spleen cells in media containing BMP4 failed to recapitulate the 45-fold increase in stress BFU-Es observed in vivo during the recovery from acute anemia. This observation suggests that additional signals present in the spleen microenvironment were also required for the expansion of stress BFU-Es.Stem cell factor (SCF) is known to play a key role in the development and expansion of erythroid progenitors. [2][3][4] SCF is encoded by the murine Steel (Sl) locus 5-7 and its receptor; Kit is encoded by the murine dominant white spotti...

Acute anemic stress induces a systemic response designed to increase oxygen delivery to hypoxic tissues. Increased erythropoiesis is a key component of this response. Recovery from acute anemia relies on stress erythropoiesis, which is distinct from steady-state erythropoiesis. In this study we found that the bone morphogenetic protein 4-dependent (BMP4-dependent) stress erythropoiesis pathway was required and specific for erythroid short-term radioprotection following bone marrow transplantation. BMP4 signaling promoted the development of three populations of stress erythroid progenitors, which expanded in the spleen subsequent to bone marrow transplantation in mice. These progenitors did not correspond to previously identified bone marrow steady-state progenitors. The most immature population of stress progenitors was capable of self renewal while maintaining erythropoiesis without contribution to other lineages when serially transplanted into irradiated secondary and tertiary recipients. These data suggest that during the immediate posttransplant period, the microenvironment of the spleen is altered, which allows donor bone marrow cells to adopt a stress erythropoietic fate and promotes the rapid expansion and differentiation of stress erythroid progenitors. Our results also suggest that stress erythropoiesis may be manipulated through targeting the BMP4 signaling pathway to improve survival after injury.

Inflammation alters bone marrow hematopoiesis to favor the production of innate immune effector cells at the expense of lymphoid cells and erythrocytes. Furthermore, proinflammatory cytokines inhibit steady-state erythropoiesis, which leads to the development of anemia in diseases with chronic inflammation. Acute anemia or hypoxic stress induces stress erythropoiesis, which generates a wave of new erythrocytes to maintain erythroid homeostasis until steady-state erythropoiesis can resume. Although hypoxia-dependent signaling is a key component of stress erythropoiesis, we found that inflammation also induced stress erythropoiesis in the absence of hypoxia. Using a mouse model of sterile inflammation, we demonstrated that signaling through Toll-like receptors (TLRs) paradoxically increased the phagocytosis of erythrocytes (erythrophagocytosis) by macrophages in the spleen, which enabled expression of the heme-responsive gene encoding the transcription factor SPI-C. Increased amounts of SPI-C coupled with TLR signaling promoted the expression ofGdf15andBmp4, both of which encode ligands that initiate the expansion of stress erythroid progenitors (SEPs) in the spleen. Furthermore, despite their inhibition of steady-state erythropoiesis in the bone marrow, the proinflammatory cytokines TNF-α and IL-1β promoted the expansion and differentiation of SEPs in the spleen. These data suggest that inflammatory signals induce stress erythropoiesis to maintain erythroid homeostasis when inflammation inhibits steady-state erythropoiesis.