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...