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...

The majority of B lymphocytes in the adult mouse are generated in the bone marrow from hematopoietic stem cells (HSCs) that first appear in the aorta-gonado-mesonephros region of the fetus on embryonic day (E) 10.5-11. Comparatively less is known about B-cell development during embryogenesis. For example, which specific embryonic tissues participate in B lymphopoiesis and whether hematopoietic differentiation is skewed toward specific B-cell subsets in the embryo are unanswered questions, because the systemic circulation is initiated early during embryogenesis, resulting in an admixture of cells potentially originating from multiple sites. We demonstrate, using Ncx1 −/− mice that lack systemic blood circulation, that the E9 yolk sac (YS) and the intra-embryonic para-aortic splanchnopleura (P-Sp) tissues independently give rise to AA4.1lo-neg B progenitor cells that preferentially differentiate into innate type B-1 and marginal zone (MZ) B cells but not into B-2 cells upon transplantation. We have further demonstrated that these B-1 progenitor cells arise directly from YS and P-Sp hemogenic endothelium. These results document the initial wave of innate B lymphopoietic progenitor cells available for seeding the fetal liver at E11. The results of these studies expand our knowledge of hemogenic endothelial sites specifying distinct B-1 and MZ cell fates apart from B-2 cells and independent of an HSC origin during development.B-1 cell | marginal zone B cell | OP9 stromal cells | hematopoiesis

The extra-embryonic yolk sac (YS) is the first hematopoietic site in the mouse embryo and is thought to generate only primitive erythroid and myeloerythroid progenitor cells before definitive HSC emergence within the embryo on E10.5. Here, we have shown the existence of T cell-restricted progenitors in the E9.5 YS that directly engraft in recipient immunodeficient mice. T-cell progenitors were also produced in vitro from both YS and para-aortic splanchnopleura hemogenic endothelial cells, and these T-cell progenitors repopulated the thymus and differentiated into mature T-cell subsets in vivo on transplantation. Our data confirm that the YS produces T-lineage-restricted progenitors that are available to colonize the thymus and provide new insight into the YS as a definitive hematopoietic site in the mouse embryo. (Blood. 2012;119(24): 5706-5714) IntroductionEmbryonic stem (ES) or induced pluripotent stem (iPS) cells have been intensively studied to understand the mechanisms regulating stem cell self-renewal and cell lineage specification and differentiation. Because ES-cell differentiation into the hematopoietic lineage mirrors the earliest aspects of normal embryonic development, 1 it is important to understand the process of developmental hematopoiesis to anticipate the products of ES or iPS differentiation. The first blood progenitor cells appear in the extra-embryonic yolk sac (YS) on E7.0. 2 These nucleated red blood cells express embryonic hemoglobin molecules and are called primitive erythroid progenitors. On E8.25, erythroid progenitor cells that express adult-type hemoglobin molecules appear in the YS and are called definitive erythroid progenitors. Likewise, primitive and definitive myeloid cells and megakaryocytes emerge in distinct waves in the YS. 3,4 HSCs, which reconstitute all the blood cell lineages that arise in adult mouse BM emerge at E10.5 in the ventral endothelial lining of the aorta in the aorta-gonad-mesonephros (AGM) region, 5,6 followed soon after on E11 in the YS, fetal liver, and placenta. 7,8 Later in development, HSCs accumulate in the fetal liver before mobilization and emigration into the BM just before birth. In adult mice, medullary HSCs self-renew and provide homeostatic blood cell production throughout life.T lymphocytes are produced and matured in the thymus, but, because there are no self-renewing stem cells in the thymus, T lymphopoiesis depends on circulating progenitor cells to continuously replenish the organ with precursors. Which BM hematopoietic progenitors colonize the adult thymus has long been controversial, but recent reports suggest that early T-lineage progenitors (lin Ϫ CD44 ϩ CD25 Ϫ CD117 ϩ IL-7R␣ loϪneg ) are the most immature T-cell progenitors found in the murine thymus. 9 Fetal T lymphopoiesis is also initiated by the colonization of extrathymic progenitor cells into the thymic anlage at E11. 10 The site and tissue of T-lymphoid progenitor emergence remains obscure. Interestingly, T, B, and myeloid lineage-committed progenitor cells, as well as multipoten...

The production of mature cells necessitates that lineage-committed progenitor cells be constantly generated from multipotential progenitors. In addition, the ability to respond rapidly to physiologic stresses requires that the signals that regulate the maintenance of progenitor populations be coordinated with the signals that promote differentiation of progenitors. Here we examine the signals that are necessary for the maintenance of the BMP4-dependent stress erythropoiesis pathway. Our previous work demonstrated that BMP4, stem cell factor, and hypoxia act in concert to promote the expansion of a specialized population of stress erythroid progenitors in the spleen during the recovery from acute anemia. Our analysis shows that acute anemia leads to an almost complete mobilization of BMP4-responsive stress erythroid burst-forming units; therefore, new stress progenitors must be recruited to the spleen to replenish this system. We show that bone marrow cells can home to the spleen and, in response to a signal in the spleen microenvironment, Hedgehog, they develop into BMP4-responsive stress progenitors. Hedgehog induces the expression of BMP4, and together these 2 signals are required for the development of BMP4-responsive stress progenitors. These data demonstrate that the interplay between these 2 signals is crucial for maintenance of this stress response pathway. (Blood. 2009;113:911-918)

The rapid growth of the embryo places severe demands on the ability of the cardiovascular system to deliver oxygen to cells. To meet this need, erythroid progenitors rapidly expand in the fetal liver microenvironment such that by E14.5, erythropoiesis predominates in the fetal liver. In this report we show that the BMP4/Smad5 dependent stress erythropoiesis pathway plays a key role in the expansion of erythroid progenitors in the fetal liver. These data show that the fetal liver contains two populations of erythroid progenitors. One population resembles the steady state erythroid progenitors found in the adult bone marrow. While the second population exhibits the properties of stress erythroid progenitors found in adult spleen. Here we demonstrate that defects in BMP4/Smad5 signaling preferentially affect the expansion of the stress erythroid progenitors in the fetal liver leading to fetal anemia. These data suggest that steady state erythropoiesis is unable to generate sufficient erythrocytes to maintain the rapid growth of the embryo leading to the induction of the BMP4 dependent stress erythropoiesis pathway. These observations underscore the similarities between fetal erythropoiesis and stress erythropoiesis.