The enucleated definitive erythrocytes of mammals are unique in the animal kingdom. The observation that yolk sacderived primitive erythroid cells in mammals circulate as nucleated cells has led to the conjecture that they are related to the red cells of fish, amphibians, and birds that remain nucleated throughout their life span. In mice, primitive red cells express both embryonic and adult hemoglobins, whereas definitive erythroblasts accumulate only adult hemoglobins. We IntroductionIt was recognized more than 125 years ago that the mature red cells of adult vertebrates circulate either in nucleated or enucleated forms. 1 The red cells of all birds, fish, reptiles, and amphibians retain their nucleus and contain 3 filamentous systems: an actinspectrin-based membrane cytoskeleton, intermediate filaments that attach the cytoskeleton to the nuclear membrane, and a group of microtubules organized into a circumferential marginal band. 2,3 In contrast, the red cells of mammals lose intermediate filaments and microtubules during terminal differentiation and enucleate prior to entering the bloodstream. Thus, erythrocytes of adult mammals are enucleated and contain only one filamentous system, a membrane cytoskeleton.Nearly 100 years ago, examination of mammalian embryos revealed the presence of distinct nucleated and enucleated red cells. 4 The continuous circulation of small, enucleated red cells during fetal and postnatal life was termed "definitive" erythropoiesis. Definitive erythropoiesis in the fetus is preceded by a "primitive" erythroid program that is characterized by the transient circulation of large, nucleated red cells that originate extraembryonically in the yolk sac. 4,5 Because primitive erythroblasts in mammals circulate as nucleated cells and are confined to the embryo, they have been thought to share many characteristics with the nucleated red cells of nonmammalian vertebrates when compared with the enucleated definitive red cells of fetal and adult mammals. 6,7 In the mouse embryo, primitive erythroid cells begin to develop in yolk sac blood islands between embryonic days 7 and 8 (E7-8). 8,9 With the onset of cardiac contractions at early somite pair stages (E8.25), primitive erythroblasts enter the embryonic bloodstream 10,11 where they remain until E16.5 when the primitive lineage was thought to be extinguished. 12,13 Definitive erythrocytes begin to emerge from the fetal liver at E12.5 13,14 and rapidly become the predominant cell type in the circulation. Definitive red cells can be distinguished from their primitive counterparts by their smaller size and by their accumulation of adult, but not embryonic, hemoglobins. 6,13,15 In contrast, primitive erythroblasts in the mouse are large cells that accumulate both embryonic and adult hemoglobins. [15][16][17] More than 30 years ago, a population of enucleated red cells with the same hemoglobin content as primitive erythroblasts was described in the embryonic circulation of the mouse. 14 Furthermore, large enucleated red cells have been noted in t...

To better understand the relationship between the embryonic hematopoietic and vascular systems, we investigated the establishment of circulation in mouse embryos by examining the redistribution of yolk sac-derived primitive erythroblasts and definitive hematopoietic progenitors. Our studies revealed that small numbers of erythroblasts first enter the embryo proper at 4 to 8 somite pairs (sp) (embryonic day 8.25 [E8.25]), concomitant with the proposed onset of cardiac function. Hours later (E8.5), most red cells remained in the yolk sac. Although the number of red cells expanded rapidly in the embryo proper, a steady state of approximately 40% red cells was not reached until 26 to 30 sp (E10). Additionally, erythroblasts were unevenly distributed within the embryo's vasculature before 35 sp. These data suggest that fully functional circulation is established after E10. This timing correlated with vascular remodeling, suggesting that vessel arborization, smooth muscle recruitment, or both are required. We also examined the distribution of committed hematopoietic progenitors during early embryogenesis. Before E8.0, all progenitors were found in the yolk sac. When normalized to circulating erythroblasts, there was a significant enrichment (20-to 5-fold) of progenitors in the yolk sac compared with the embryo proper from E9.5 to E10.5. These results indicated that the yolk sac vascular network remains a site of progenitor production and preferential adhesion even as the fetal liver becomes a hematopoietic organ. We conclude that a functional vascular system develops gradually and that specialized vascular-hematopoietic environments exist after circulation becomes fully established. IntroductionA functional circulatory system is an early requirement for survival and growth of the mammalian embryo and is the first organ system to develop in the embryo. 1 The circulatory system is composed of vascular, hematopoietic, and cardiac components, each formed from discrete regions of mesoderm. The first endothelial cells and blood cells are generated in yolk sac blood islands beginning at embryonic day 7 (E7.0) in the mouse. By E8.0, thousands of nucleated primitive red blood cells have formed within a vascular plexus in the yolk sac. [2][3][4] Concurrently, the aorta and the peristaltic beating heart tube form in the embryo proper. In the next 36 hours, there is a remarkable increase in complexity of vascular and hematopoietic systems. The vascular plexus remodels and expands into an arborized network of specialized arteries and veins. Primitive erythroblasts from the yolk sac continue to divide and mature, and a second wave of hematopoiesis creating definitive (adultlike) progenitors originates in the yolk sac. 5,6 Thus, the early vascular system is the hematopoietic environment for primitive and definitive lineages until the specialized stromal microenvironment of the fetal liver (beginning E10), and later the adult bone marrow, is available. However, the nature of any specific interactions between early embryonic hematopo...

Key Points• Comparative global gene expression analysis of primary murine primitive, fetal definitive, and adult definitive erythroid precursors.• Primitive erythroblasts contain and accumulate high ROS levels and uniquely express the H2O2 transporting aquaporins 3 and 8.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 H 2 O 2 transporters, primitive, but not definitive, erythroblasts preferentially accumulate reactive oxygen species after exogenous H 2 O 2 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. (Blood. 2013;121(6):e5-e13) IntroductionRBCs constitute an estimated 1 in 4 cells in the body and are necessary for tissue oxygen delivery. In the adult, RBCs are produced primarily in the BM where lineage-committed progenitors give rise to morphologically identifiable precursors. Erythroid precursors physically associate with macrophages and undergo several maturational cell divisions characterized by a progressive decrease in cell size, nuclear condensation, hemoglobin accumulation, and loss of RNA content. 1 These physical changes have been used to classify erythroid precursors into proerythroblast, basophilic, polychromatophilic, and orthochromatic erythroblast maturational stages. In mammals, orthochromatic erythroblasts enucleate to form reticulocytes that ultimately enter the circulation and complete their maturation.Erythroid cells are a critical component of the cardiovascular network, which constitutes the first functional organ system in the mammalian embryo. 2 "Primitive" erythroid cells first emerge in yolk sac blood islands. 3 We previously determined that primitive erythroid cells originate from a transient wave of committed progenitors in the yolk sac and mature as a semisynchronous cohort in the bloodstream, undergoing morphologic changes similar to those observed in defini...

Mammals have 2 distinct erythroid lineages. The primitive erythroid lineage originates in the yolk sac and generates a cohort of large erythroblasts that terminally differentiate in the bloodstream. The definitive erythroid lineage generates smaller enucleated erythrocytes that become the predominant cell in fetal and postnatal circulation. These lineages also have distinct globin expression patterns. Our studies in primary murine primitive erythroid cells indicate that ␤H1 is the predominant ␤-globin transcript in the early yolk sac. Thus, unlike the human, murine ␤-globin genes are not up-regulated in the order of their chromosomal arrangement. As primitive erythroblasts mature from proerythroblasts to reticulocytes, they undergo a ␤H1-to ⑀y-globin switch, up-regulate adult ␤1-and ␤2-globins, and down-regulate -globin. These changes in transcript levels correlate with changes in RNA polymerase II density at their promoters and transcribed regions. Furthermore, the ⑀y-and ␤H1-globin genes in primitive erythroblasts reside within a single large hyperacetylated domain. These data suggest that this "maturational" ␤H1-to ⑀y-globin switch is dynamically regulated at the transcriptional level. Globin switching during ontogeny is due not only to the sequential appearance of primitive and definitive lineages but also to changes in globin expression as primitive erythroblasts mature in the bloodstream. IntroductionThe first blood cells to circulate during mammalian embryogenesis consist of large primitive red cells. In the mouse embryo, primitive erythroid cells are generated from a transient wave of committed progenitors found in the yolk sac between embryonic day (E) 7.25 and E9.0 of gestation. 1,2 Primitive proerythroblasts begin to emerge from blood islands beginning at E8.25, and enter the newly forming bloodstream, 3 where they undergo terminal maturation. They transition as a semisynchronous cohort of cells from proerythroblasts to enucleated erythrocytes progressively undergoing a loss of proliferative capacity, a decrease in cell size, accumulation of hemoglobin, and nuclear condensation. [4][5][6] Ultimately, they enucleate by E16.5 to become primitive erythrocytes that can circulate for several days after birth. 7 After E11.5, primitive red cells are joined by increasing numbers of smaller definitive erythrocytes that are released from the fetal liver after enucleating. Over the ensuing 5 days, definitive erythrocytes become the predominant cell type in the fetal circulation and ultimately become the exclusive red-cell lineage in the adult.The sine qua non of red cells is their accumulation of large amounts of hemoglobin composed of globin tetramers encoded by the ␣-and ␤-globin gene loci. Examination of globin expression in circulating human and mouse blood cells indicated that 5Ј members of both globin loci are expressed in the embryo, while 3Ј members are expressed in the adult, leading to the concept of globin switching during ontogeny. 8 Since this switch in globin expression coincided temporally with t...

Primitive erythroid cells, the first red blood cells produced in the mammalian embryo, are necessary for embryonic survival. Erythropoietin and its receptor EpoR, are absolutely required for survival of late-stage definitive erythroid progenitors in the fetal liver and adult bone marrow. Epo-and Epor-null mice die at E13.5 with a lack of definitive erythrocytes. However, the persistence of circulating primitive erythroblasts raises questions about the role of erythropoietin/EpoR in primitive erythropoiesis. Using Epor-null mice and a novel primitive erythroid 2-step culture we found that erythropoietin is not necessary for specification of primitive erythroid progenitors. However, Epornull embryos develop a progressive, profound anemia by E12.5 as primitive erythroblasts mature as a synchronous cohort. This anemia results from reduced primitive erythroblast proliferation associated with increased p27 expression, from advanced cellular maturation, and from markedly elevated rates of apoptosis associated with an imbalance in pro-and anti-apoptotic gene expression. Both mouse and human primitive erythroblasts cultured without erythropoietin also undergo accelerated maturation and apoptosis at later stages of maturation. We conclude that erythropoietin plays an evolutionarily conserved role in promoting the proliferation, survival, and appropriate timing of terminal maturation of primitive erythroid precursors. Erythropoietin critically regulates the terminal maturation of murine and human primitive erythroblasts

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.

The normal counterparts of mantle cell lymphoma (MCL) are naïve quiescent B-cells that have not been processed through the germinal center (GC). For this reason, while lymphomas arising from GC or post-GC B-cells often exhibit plasmacytic differentiation, MCL rarely presents with plasmacytic features. Seven cases of MCL with a monotypic plasma cell (PC) population were collected from six centers and studied by immunohistochemistry, FICTION (Fluorescence immunophenotyping and Interphase Cytogenetics as a Tool for the Investigation of Neoplasms), capillary gel electrophoresis, and restriction fragment length polymorphism of immunoglobulin heavy chain analysis (RFLP/IgH) of microdissections of each of the MCL and PC populations to assess their clonal relationship. Clinical presentation was rather unusual compared to typical MCL, with two cases arising from extranodal soft-tissues of the head. All MCL cases were morphologically and immunohistochemically typical, bearing the t(11;14)(q13;q32). In all cases PC populations were clonal. In 5 of the 7 cases, the MCL and PC clones showed identical restriction fragments, indicating a common clonal origin of the neoplastic populations. The two cases with clonal diversity denoted the coexistence of two different tumors in a composite lymphoma/plasma cell neoplasm. Our findings suggest that MCL can present with a PC component that is often clonally related to the lymphoma, representing a rare but unique biological variant of this tumor.

BackgroundHematopoietic ontogeny is characterized by overlapping waves of primitive, fetal definitive, and adult definitive erythroid lineages. Our aim is to identify differences in the transcriptional control of these distinct erythroid cell maturation pathways by inferring and analyzing gene-interaction networks from lineage-specific expression datasets. Inferred networks are strongly connected and do not fit a scale-free model, making it difficult to identify essential regulators using the hub-essentiality standard.ResultsWe employed a semi-supervised machine learning approach to integrate measures of network topology with expression data to score gene essentiality. The algorithm was trained and tested on the adult and fetal definitive erythroid lineages. When applied to the primitive erythroid lineage, 144 high scoring transcription factors were found to be differentially expressed between the primitive and adult definitive erythroid lineages, including all expressed STAT-family members. Differential responses of primitive and definitive erythroblasts to a Stat3 inhibitor and IFNγ in vitro supported the results of the computational analysis. Further investigation of the original expression data revealed a striking signature of Stat1-related genes in the adult definitive erythroid network. Among the potential pathways known to utilize Stat1, interferon (IFN) signaling-related genes were expressed almost exclusively within the adult definitive erythroid network.ConclusionsIn vitro results support the computational prediction that differential regulation and downstream effectors of STAT signaling are key factors that distinguish the transcriptional control of primitive and definitive erythroid cell maturation.