Endoscopic Septoplasty: A Retrospective Analysis of Indications and Outcome.

Background: Endoscopic septoplasty is a minimally invasive surgical procedure for the correction of nasal septal deformity. Globally, nasal septal surgeries are rarely performed, and in our country these procedures are even more scarcely undertaken, partly due to dearth of facilities and to some extent, the expertise of embarking on this specialised surgical procedure. Therefore, we aimed to document the indications and the outcome of endoscopic septoplasty in our environment. Materials and Methods: This was a retrospective study of all consecutive patients that had endoscopic septoplasty at a state tertiary hospital over three years period. Ethical approval was obtained before commencement of the study. Patients' medical records were retrieved. Biodata, clinical presentation, operative procedure and outcome were extracted and analyzed descriptively. Results: Fourteen patients had endoscopic septoplasty over the period under review, constituting 11 (78.6%) males and 3 (21.4%) females. Predominant clinical features were nasal obstruction (100%) and nasal septal deviation (100%). The main indication for procedure was deviated nasal septum. The outcome of the surgery was good, 2(14.3%) of the patients had nasal adhesions but no major complication was recorded. The length of hospital stay ranged between 3 and 5 days with a mean of 3.7 ± 0.9 days, and all the patients were discharged successfully. Conclusions: Endoscopic septoplasty is a safe surgery. The main indication for the procedure was deviated nasal septum, and the procedure has a favourable outcome among the operated patients.

Mds1CreERT2, an inducible Cre allele specific to adult-repopulating hematopoietic stem cells.

Hematopoietic ontogeny consists of two broad programs: an initial hematopoietic stem cell (HSC)-independent program followed by HSC-dependent hematopoiesis that sequentially seed the fetal liver and generate blood cells. However, the transition from HSC-independent to HSC-derived hematopoiesis remains poorly characterized. To help resolve this question, we developed Mds1CreERT2 mice, which inducibly express Cre-recombinase in emerging HSCs in the aorta and label long-term adult HSCs, but not HSC-independent yolk-sac-derived primitive or definitive erythromyeloid (EMP) hematopoiesis. Our lineage-tracing studies indicate that HSC-derived erythroid, myeloid, and lymphoid progeny significantly expand in the liver and blood stream between E14.5 and E16.5. Additionally, we find that HSCs contribute the majority of F4/80+ macrophages in adult spleen and marrow, in contrast to their limited contribution to macrophage populations in brain, liver, and lungs. The Mds1CreERT2 mouse model will be useful to deconvolute the complexity of hematopoiesis as it unfolds in the embryo and functions postnatally.

Hyperacetylated chromatin domains mark cell type-specific genes and suggest distinct modes of enhancer function.

Stratification of enhancers by signal strength in ChIP-seq assays has resulted in the establishment of super-enhancers as a widespread and useful tool for identifying cell type-specific, highly expressed genes and associated pathways. We examine a distinct method of stratification that focuses on peak breadth, termed hyperacetylated chromatin domains (HCDs), which classifies broad regions exhibiting histone modifications associated with gene activation. We find that this analysis serves to identify genes that are both more highly expressed and more closely aligned to cell identity than super-enhancer analysis does using multiple data sets. Moreover, genetic manipulations of selected gene loci suggest that some enhancers located within HCDs work at least in part via a distinct mechanism involving the modulation of histone modifications across domains and that this activity can be imported into a heterologous gene locus. In addition, such genetic dissection reveals that the super-enhancer concept can obscure important functions of constituent elements.

Potently Cytotoxic Natural Killer Cells Initially Emerge from Erythro-Myeloid Progenitors during Mammalian Development.

Natural killer (NK) cells are a critical component of the innate immune system. However, their ontogenic origin has remained unclear. Here, we report that NK cell potential first arises from Hoxaneg/low Kit+CD41+CD16/32+ hematopoietic-stem-cell (HSC)-independent erythro-myeloid progenitors (EMPs) present in the murine yolk sac. EMP-derived NK cells and primary fetal NK cells, unlike their adult counterparts, exhibit robust degranulation in response to stimulation. Parallel studies using human pluripotent stem cells (hPSCs) revealed that HOXAneg/low CD34+ progenitors give rise to NK cells that, similar to murine EMP-derived NK cells, harbor a potent cytotoxic degranulation bias. In contrast, hPSC-derived HOXA+ CD34+ progenitors, as well as human cord blood CD34+ cells, give rise to NK cells that exhibit an attenuated degranulation response but robustly produce inflammatory cytokines. Collectively, our studies identify an extra-embryonic origin of potently cytotoxic NK cells, suggesting that ontogenic origin is a relevant factor in designing hPSC-derived adoptive immunotherapies.

Acute and late effects of combined internal and external radiation exposures on the hematopoietic system.

Purpose: Incidents, such as nuclear facility accidents and the release of a 'dirty bomb', might result in not only external irradiation of personnel, but additional internal exposures through concomitant inhalation and/or ingestion of radioactive particulates. The purpose of this study was to define the impact of such a combination of radiation injuries on the hematopoietic niche.Material and methods: To assess changes in the murine hematopoietic system, we used a combined exposure of total body irradiation (TBI, 6 Gy) followed immediately by an internal (intraperitoneal) administration of 100 µCi of soluble 137Cs. We then evaluated acute survival in combined versus single modality exposure groups, as well as assessing hematopoietic function at 12 and 26 week time points.Results: Acutely, the combination of external and internal exposures led to an unexpected delay in excretion of 137Cs, increasing the absorbed dose in the combined exposure group and leading to mortality from an acute hematopoietic syndrome. At 12 weeks, all exposure paradigms resulted in decreased numbers of phenotypic hematopoietic stem cells (HSCs), particularly the short-term HSCs (ST-HSC); long-term HSCs (LT-HSC) were depleted only in the internal and combined exposure groups. At 26 weeks, there was significant anemia in both the TBI alone and combined exposure groups. There were decreased numbers in both the LT- and ST-HSCs and decreased functionality, as measured by competitive repopulation, was seen in all radiation groups, with the greatest effects seen in the internal and combined exposure groups.Conclusions: Our data indicate that a combined injury of sublethal external irradiation with internal contamination induces significant and persistent changes in the hematopoietic system, as may have been predicted from the literature and our own group's findings. However, a novel observation was that the combined exposure led to an alteration in the excretion kinetics of the internal contamination, increasing the acute effects beyond those anticipated. As a result, we believe that a combined exposure poses a unique challenge to the medical community during both the acute and, possibly, delayed recovery stages.

Lin28b regulates age-dependent differences in murine platelet function.

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.

FAM210B is an erythropoietin target and regulates erythroid heme synthesis by controlling mitochondrial iron import and ferrochelatase activity.

Erythropoietin (EPO) signaling is critical to many processes essential to terminal erythropoiesis. Despite the centrality of iron metabolism to erythropoiesis, the mechanisms by which EPO regulates iron status are not well-understood. To this end, here we profiled gene expression in EPO-treated 32D pro-B cells and developing fetal liver erythroid cells to identify additional iron regulatory genes. We determined that FAM210B, a mitochondrial inner-membrane protein, is essential for hemoglobinization, proliferation, and enucleation during terminal erythroid maturation. Fam210b deficiency led to defects in mitochondrial iron uptake, heme synthesis, and iron-sulfur cluster formation. These defects were corrected with a lipid-soluble, small-molecule iron transporter, hinokitiol, in Fam210b-deficient murine erythroid cells and zebrafish morphants. Genetic complementation experiments revealed that FAM210B is not a mitochondrial iron transporter but is required for adequate mitochondrial iron import to sustain heme synthesis and iron-sulfur cluster formation during erythroid differentiation. FAM210B was also required for maximal ferrochelatase activity in differentiating erythroid cells. We propose that FAM210B functions as an adaptor protein that facilitates the formation of an oligomeric mitochondrial iron transport complex, required for the increase in iron acquisition for heme synthesis during terminal erythropoiesis. Collectively, our results reveal a critical mechanism by which EPO signaling regulates terminal erythropoiesis and iron metabolism.

Circulating primitive erythroblasts establish a functional, protein 4.1R-dependent cytoskeletal network prior to enucleating.

Hematopoietic ontogeny is characterized by distinct primitive and definitive erythroid lineages. Definitive erythroblasts mature and enucleate extravascularly and form a unique membrane skeleton, composed of spectrin, 4.1R-complex, and ankyrinR-complex components, to survive the vicissitudes of the adult circulation. However, little is known about the formation and composition of the membrane skeleton in primitive erythroblasts, which progressively mature while circulating in the embryonic bloodstream. We found that primary primitive erythroblasts express the major membrane skeleton genes present in similarly staged definitive erythroblasts, suggesting that the composition and formation of this membrane network is conserved in maturing primitive and definitive erythroblasts despite their respective intravascular and extravascular locations. Membrane deformability and stability of primitive erythroblasts, assayed by microfluidic studies and fluorescence imaged microdeformation, respectively, significantly increase prior to enucleation. These functional changes coincide with protein 4.1 R isoform switching and protein 4.1R-null primitive erythroblasts fail to establish normal membrane stability and deformability. We conclude that maturing primitive erythroblasts initially navigate the embryonic vasculature prior to establishing a deformable cytoskeleton, which is ultimately formed prior to enucleation. Formation of an erythroid-specific, protein 4.1R-dependent membrane skeleton is an important feature not only of definitive, but also of primitive, erythropoiesis in mammals.

Adenosine-to-inosine RNA editing by ADAR1 is essential for normal murine erythropoiesis.

Adenosine deaminases that act on RNA (ADARs) convert adenosine residues to inosine in double-stranded RNA. In vivo, ADAR1 is essential for the maintenance of hematopoietic stem/progenitors. Whether other hematopoietic cell types also require ADAR1 has not been assessed. Using erythroid- and myeloid-restricted deletion of Adar1, we demonstrate that ADAR1 is dispensable for myelopoiesis but is essential for normal erythropoiesis. Adar1-deficient erythroid cells display a profound activation of innate immune signaling and high levels of cell death. No changes in microRNA levels were found in ADAR1-deficient erythroid cells. Using an editing-deficient allele, we demonstrate that RNA editing is the essential function of ADAR1 during erythropoiesis. Mapping of adenosine-to-inosine editing in purified erythroid cells identified clusters of hyperedited adenosines located in long 3'-untranslated regions of erythroid-specific transcripts and these are ADAR1-specific editing events. ADAR1-mediated RNA editing is essential for normal erythropoiesis.

Bmi-1 Regulates Extensive Erythroid Self-Renewal.

Red 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.

Distinct Sources of Hematopoietic Progenitors Emerge before HSCs and Provide Functional Blood Cells in the Mammalian Embryo.

Hematopoietic potential arises in mammalian embryos before adult-repopulating hematopoietic stem cells (HSCs). At embryonic day 9.5 (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. Although 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. Furthermore, we find that embryonic stem cell (ESC)-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 they can provide transient adult-like RBC reconstitution.

The mTORC1/4E-BP pathway coordinates hemoglobin production with L-leucine availability.

In multicellular organisms, the mechanisms by which diverse cell types acquire distinct amino acids and how cellular function adapts to their availability are fundamental questions in biology. We found that increased neutral essential amino acid (NEAA) uptake was a critical component of erythropoiesis. As red blood cells matured, expression of the amino acid transporter gene Lat3 increased, which increased NEAA import. Inadequate NEAA uptake by pharmacologic inhibition or RNAi-mediated knockdown of LAT3 triggered a specific reduction in hemoglobin production in zebrafish embryos and murine erythroid cells through the mTORC1 (mammalian target of rapamycin complex 1)/4E-BP (eukaryotic translation initiation factor 4E-binding protein) pathway. CRISPR-mediated deletion of members of the 4E-BP family in murine erythroid cells rendered them resistant to mTORC1 and LAT3 inhibition and restored hemoglobin production. These results identify a developmental role for LAT3 in red blood cells and demonstrate that mTORC1 serves as a homeostatic sensor that couples hemoglobin production at the translational level to sufficient uptake of NEAAs, particularly L-leucine.

TMEM14C is required for erythroid mitochondrial heme metabolism.

The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.

SDF-1 dynamically mediates megakaryocyte niche occupancy and thrombopoiesis at steady state and following radiation injury.

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 cell-derived 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 MK-vasculature 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.

Snx3 regulates recycling of the transferrin receptor and iron assimilation.

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.

Ontogeny of erythroid gene expression.

Erythroid ontogeny is characterized by overlapping waves of primitive and definitive erythroid lineages that share many morphologic features during terminal maturation but have marked differences in cell size and globin expression. In the present study, we compared global gene expression in primitive, fetal definitive, and adult definitive erythroid cells at morphologically equivalent stages of maturation purified from embryonic, fetal, and adult mice. Surprisingly, most transcriptional complexity in erythroid precursors is already present by the proerythroblast stage. Transcript levels are markedly modulated during terminal erythroid maturation, but housekeeping genes are not preferentially lost. Although primitive and definitive erythroid lineages share a large set of nonhousekeeping genes, annotation of lineage-restricted genes shows that alternate gene usage occurs within shared functional categories, as exemplified by the selective expression of aquaporins 3 and 8 in primitive erythroblasts and aquaporins 1 and 9 in adult definitive erythroblasts. Consistent with the known functions of Aqp3 and Aqp8 as H2O2 transporters, primitive, but not definitive, erythroblasts preferentially accumulate reactive oxygen species after exogenous H2O2 exposure. We have created a user-friendly Web site (http://www.cbil.upenn.edu/ErythronDB) to make these global expression data readily accessible and amenable to complex search strategies by the scientific community.

EPO-mediated expansion of late-stage erythroid progenitors in the bone marrow initiates recovery from sublethal radiation stress.

Erythropoiesis is a robust process of cellular expansion and maturation occurring in murine bone marrow and spleen. We previously determined that sublethal irradiation, unlike bleeding or hemolysis, depletes almost all marrow and splenic erythroblasts but leaves peripheral erythrocytes intact. To better understand the erythroid stress response, we analyzed progenitor, precursor, and peripheral blood compartments of mice post-4 Gy total body irradiation. Erythroid recovery initiates with rapid expansion of late-stage erythroid progenitors-day 3 burst-forming units and colony-forming units, associated with markedly increased plasma erythropoietin (EPO). Although initial expansion of late-stage erythroid progenitors is dependent on EPO, this cellular compartment becomes sharply down-regulated despite elevated EPO levels. Loss of EPO-responsive progenitors is associated temporally with a wave of maturing erythroid precursors in marrow and with emergence of circulating erythroid progenitors and subsequent reestablishment of splenic erythropoiesis. These circulating progenitors selectively engraft and mature in irradiated spleen after short-term transplantation, supporting the concept that bone marrow erythroid progenitors migrate to spleen. We conclude that sublethal radiation is a unique model of endogenous stress erythropoiesis, with specific injury to the extravascular erythron, expansion and maturation of EPO-responsive late-stage progenitors exclusively in marrow, and subsequent reseeding of extramedullary sites.

A transient definitive erythroid lineage with unique regulation of the ß-globin locus in the mammalian embryo.

A transient erythromyeloid wave of definitive hematopoietic progenitors (erythroid/myeloid progenitors [EMPs]) emerges in the yolk sac beginning at embryonic day 8.25 (E8.25) and colonizes the liver by E10.5, before adult-repopulating hematopoietic stem cells. At E11.5, we observe all maturational stages of erythroid precursors in the liver and the first definitive erythrocytes in the circulation. These early fetal liver erythroblasts express predominantly adult ß-globins and the definitive erythroid-specific transcriptional modifiers c-myb, Sox6, and Bcl11A. Surprisingly, they also express low levels of "embryonic" ßH1-, but not εy-, globin transcripts. Consistent with these results, RNA polymerase and highly modified histones are found associated with ßH1- and adult globin, but not εy-globin, genes. E11.5 definitive proerythroblasts from mice transgenic for the human ß-globin locus, like human fetal erythroblasts, express predominately human γ-, low ß-, and no ε-globin transcripts. Significantly, E9.5 yolk sac-derived EMPs cultured in vitro have similar murine and human transgenic globin expression patterns. Later liver proerythroblasts express low levels of γ-globin, while adult marrow proerythroblasts express only ß-globin transcripts. We conclude that yolk sac-derived EMPs, the first of 2 origins of definitive erythropoiesis, express a unique pattern of globin genes as they generate the first definitive erythrocytes in the liver of the mammalian embryo.

Sublethal radiation injury uncovers a functional transition during erythroid maturation.

OBJECTIVE: Clastogenic injury of the erythroid lineage results in anemia, reticulocytopenia, and transient appearance of micronucleated reticulocytes. However, the micronucleated reticulocyte dose-response in murine models is only linear to 2 Gy total body irradiation and paradoxically decreases at higher exposures, suggesting complex radiation effects on erythroid intermediates. To better understand this phenomenon, we investigated the kinetics and apoptotic response of the erythron to sublethal radiation injury. MATERIALS AND METHODS: We analyzed the response to 1 and 4 Gy total body irradiation of erythroid progenitors and precursors using colony assays and imaging flow cytometry, respectively. We also investigated cell cycling and apoptotic gene expression of the steady-state erythron. RESULTS: After 1 Gy total body irradiation, erythroid progenitors and precursors were partially depleted. In contrast, essentially all bone marrow erythroid progenitors and precursors were lost within 2 days after 4 Gy irradiation. Imaging flow cytometry analysis revealed preferential loss of phenotypic erythroid colony-forming units and proerythroblasts immediately after sublethal irradiation. Furthermore, these populations underwent radiation-induced apoptosis, without changes in steady-state cellular proliferation, at much higher frequencies than later-stage erythroid precursors. Primary erythroid precursor maturation is associated with marked Bcl-xL upregulation and Bax and Bid downregulation. CONCLUSIONS: Micronucleated reticulocyte loss after higher sublethal radiation exposures results from rapid depletion of erythroid progenitors and precursors. This injury reveals that erythroid colony-forming units and proerythroblasts constitute a particularly proapoptotic compartment within the erythron. We conclude that the functional transition of primary proerythroblasts to later-stage erythroid precursors is characterized by a shift from a proapoptotic to an antiapoptotic phenotype.

Primitive erythropoiesis in the mammalian embryo.

Erythropoiesis in adult mammals is characterized by the progressive maturation of hematopoietic stem cells to lineage-specific progenitors, to morphologically identifiable precursors which enucleate to form mature erythrocytes. In contrast, primitive erythropoiesis is characterized by the appearance within the yolk sac of a transient, lineage-restricted progenitor population which generates a wave of erythroid precursors. These precursors undergo progressive maturation in the bloodstream, characterized by nuclear condensation and embryonic hemoglobin accumulation. This process is dependent on erythropoietin signaling through its cognate receptor, as well as the function of several erythroid-specific transcription factors, including GATA1 and EKLF. Targeted disruption of genes in the mouse that result in failure of the emergence or maturation of the primitive erythroid lineage leads to early fetal death, indicating that the primitive erythroid lineage is necessary for survival of the mammalian embryo. While it was thought for over a century that primitive erythroid cells were uniquely nucleated mammalian red cells, it is now recognized that they, like their definitive erythroid counterparts, enucleate to form reticulocytes and pyrenocytes. This surprising finding indicates that the primitive erythroid lineage is indeed mammalian, rather than non-mammalian, in character.