Summary Hematopoietic potential arises in mammalian embryos before adult-repopulating hematopoietic stem cells (HSCs). At E9.5, we show the first murine definitive erythro-myeloid progenitors (EMPs) have an immunophenotype distinct from primitive hematopoietic progenitors, maturing megakaryocytes and macrophages, and rare B cell potential. EMPs emerge in the yolk sac with erythroid and broad myeloid, but not lymphoid, potential. EMPs migrate to the fetal liver and rapidly differentiate including production of circulating neutrophils by E11.5. While the surface markers, transcription factors and lineage potential associated with EMPs overlap with those found in adult definitive hematopoiesis, they are present in unique combinations or proportions that result in a specialized definitive embryonic progenitor. Further, we find that ES cell -derived hematopoiesis recapitulates early yolk sac hematopoiesis, including primitive, EMP and rare B cell potential. EMPs do not have long term potential when transplanted in immunocompromised adults, but can provide transient adult-like RBC reconstitution.

Adult-repopulating hematopoietic stem cells (HSCs) emerge in low numbers in the midgestation mouse embryo from a subset of arterial endothelium, through an endothelial-to-hematopoietic transition. HSC-producing arterial hemogenic endothelium relies on the establishment of embryonic blood flow and arterial identity, and requires β-catenin signaling. Specified prior to and during the formation of these initial HSCs are thousands of yolk sac-derived erythro-myeloid progenitors (EMPs). EMPs ensure embryonic survival prior to the establishment of a permanent hematopoietic system, and provide subsets of long-lived tissue macrophages. While an endothelial origin for these HSC-independent definitive progenitors is also accepted, the spatial location and temporal output of yolk sac hemogenic endothelium over developmental time remains undefined. We performed a spatiotemporal analysis of EMP emergence, and document the morphological steps of the endothelial-to-hematopoietic transition. Emergence of rounded EMPs from polygonal clusters of Kit+ cells initiates prior to the establishment of arborized arterial and venous vasculature in the yolk sac. Interestingly, Kit+ polygonal clusters are detected in both arterial and venous vessels after remodeling. To determine whether there are similar mechanisms regulating the specification of EMPs with other angiogenic signals regulating adult-repopulating HSCs, we investigated the role of embryonic blood flow and Wnt/β-catenin signaling during EMP emergence. In embryos lacking a functional circulation, rounded Kit+ EMPs still fully emerge from unremodeled yolk sac vasculature. In contrast, canonical Wnt signaling appears to be a common mechanism regulating hematopoietic emergence from hemogenic endothelium. These data illustrate the heterogeneity in hematopoietic output and spatiotemporal regulation of primary embryonic hemogenic endothelium.

SUMMARY Sorting of endocytic ligands and receptors is critical for diverse cellular processes. The physiological significance of endosomal sorting proteins in vertebrates, however, remains largely unknown. Here we report that sorting nexin 3 (Snx3) facilitates the recycling of transferrin receptor (Tfrc), and thus is required for the proper delivery of iron to erythroid progenitors. Snx3 is highly expressed in vertebrate hematopoietic tissues. Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates due to impaired transferrin (Tf)-mediated iron uptake and its accumulation in early endosomes. This impaired iron assimilation can be complemented with non-Tf iron chelates. We show that Snx3 and Vps35, a component of the retromer, interact with Tfrc to sort it to the recycling endosomes. Our findings uncover a role of Snx3 in regulating Tfrc recycling, iron homeostasis, and erythropoiesis. Thus, the identification of Snx3 provides a genetic tool for exploring erythropoiesis and disorders of iron metabolism.

Highlights d NK cell potential arises from erythro-myeloid progenitors (EMPs) in the yolk sac d EMP-derived NK cells, similar to fetal NK cells, have a potent degranulation response d hPSC differentiation yields 2 distinct CD34 + populations, each with NK cell potential d hPSC-derived EMP-like NK cells are more potently cytotoxic than adult CD16 + NK cells

• SDF-1 acutely affects megakaryocyte spatial distribution in the bone marrow at steady state and in the setting of radiation injury.• SDF-1-directed localization of megakaryocytes into the vascular niche increases platelet output.Megakaryocyte (MK) development in the bone marrow progresses spatially from the endosteal niche, which promotes MK progenitor proliferation, to the sinusoidal vascular niche, the site of terminal maturation and thrombopoiesis. The chemokine stromal cellderived factor-1 (SDF-1), signaling through CXCR4, is implicated in the maturational chemotaxis of MKs toward sinusoidal vessels. Here, we demonstrate that both IV administration of SDF-1 and stabilization of endogenous SDF-1 acutely increase MKvasculature association and thrombopoiesis with no change in MK number. In the setting of radiation injury, we find dynamic fluctuations in marrow SDF-1 distribution that spatially and temporally correlate with variations in MK niche occupancy. Stabilization of altered SDF-1 gradients directly affects MK location. Importantly, these SDF-1-mediated changes have functional consequences for platelet production, as the movement of MKs away from the vasculature decreases circulating platelets, while MK association with the vasculature increases circulating platelets. Finally, we demonstrate that manipulation of SDF-1 gradients can improve radiation-induced thrombocytopenia in a manner additive with earlier TPO treatment. Taken together, our data support the concept that SDF-1 regulates the spatial distribution of MKs in the marrow and consequently circulating platelet numbers. This knowledge of the microenvironmental regulation of the MK lineage could lead to improved therapeutic strategies for thrombocytopenia. (Blood. 2014;124(2):277-286) Introduction Platelet-producing megakaryocytes (MKs) are derived from megakaryocyte progenitors (MKPs), which are defined functionally by their capacity to form colonies in vitro. 1,2 MKPs are thought to reside near the bone surface in an "endosteal niche," where environmental cues encourage expansion, but suppress terminal maturation.3-6 Polyploid MKs mature cytoplasmically, extrude proplatelets in association with sinusoidal vasculature, and shed platelets into the peripheral blood. [7][8][9] This process results in past-maturity "exhausted" MKs comprised of a nucleus with a thin layer of cytoplasm surrounded by a cell membrane. 10,11 Megakaryopoiesis is primarily regulated by the cytokine thrombopoietin (TPO), which signals through its receptor Mpl to promote MKP proliferation and MK maturation. [12][13][14] Although the physical association of MKs with sinusoidal vasculature was first appreciated several decades ago, 15-17 the functional significance of the "vascular niche" for MK maturation and thrombopoiesis has only more recently begun to be elucidated. 4,[18][19][20][21] Several studies have implicated the chemokine stromal cellderived factor-1 ([SDF-1] or CXCL12) signaling through receptor CXCR4 in the maturational localization of MKs to the vascular nic...

SummaryRed blood cells (RBCs), responsible for oxygen delivery and carbon dioxide exchange, are essential for our well-being. Alternative RBC sources are needed to meet the increased demand for RBC transfusions projected to occur as our population ages. We previously have discovered that erythroblasts derived from the early mouse embryo can self-renew extensively ex vivo for many months. To better understand the mechanisms regulating extensive erythroid self-renewal, global gene expression data sets from self-renewing and differentiating erythroblasts were analyzed and revealed the differential expression of Bmi-1. Bmi-1 overexpression conferred extensive self-renewal capacity upon adult bone-marrow-derived self-renewing erythroblasts, which normally have limited proliferative potential. Importantly, Bmi-1 transduction did not interfere with the ability of extensively self-renewing erythroblasts (ESREs) to terminally mature either in vitro or in vivo. Bmi-1-induced ESREs can serve to generate in vitro models of erythroid-intrinsic disorders and ultimately may serve as a source of cultured RBCs for transfusion therapy.

Platelets are essential for hemostasis; however, several studies have identified age-dependent differences in platelet function. To better understand the origins of fetal platelet function, we have evaluated the contribution of the fetal-specific RNA binding protein Lin28b in the megakaryocyte/platelet lineage. Because activated fetal platelets have very low levels of P-selectin, we hypothesized that the expression of platelet P-selectin is part of a fetal-specific hematopoietic program conferred by Lin28b. Using the mouse as a model, we find that activated fetal platelets have low levels of P-selectin and do not readily associate with granulocytes in vitro and in vivo, relative to adult controls. Transcriptional analysis revealed high levels of Lin28b and Hmga2 in fetal, but not adult, megakaryocytes. Overexpression of LIN28B in adult mice significantly reduces the expression of P-selectin in platelets, and therefore identifies Lin28b as a negative regulator of P-selectin expression. Transplantation of fetal hematopoietic progenitors resulted in the production of platelets with low levels of P-selectin, suggesting that the developmental regulation of P-selectin is intrinsic and independent of differences between fetal and adult microenvironments. Last, we observe that the upregulation of P-selectin expression occurs postnatally, and the temporal kinetics of this upregulation are recapitulated by transplantation of fetal hematopoietic stem and progenitor cells into adult recipients. Taken together, these studies identify Lin28b as a new intrinsic regulator of fetal platelet function.

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