Ontogeny of the antibody-forming cell line in mice. IV. Appearance of cells bearing Fc receptors, complement receptors, and surface immunoglobulin.

The ontogeny of Ig, FcR, and CR-bearing cells in liver and spleen has been followed by using rosetting procedures. These studies demonstrated a sequential appearance of surface receptors during development. Two types of Ig+ cells could be distinguished according to their rosette morphology and adherence to carbonyl iron: 1) an adherent cell which bound few erythrocytes was found predominantly in fetal liver from 13 days gestation and 2) a nonadherent cell which bound larger numbers of erythrocytes appeared in small numbers in fetal liver from day-16 gestation but represented the major Ig+ cell type after birth. Changes in the proportions of receptor-bearing populations occurred at two particular periods during ontogeny. The first was at birth, where an increase in the proportion of FcR+ cells occurred and the proportion of type 2 Ig+ cells rose rapidly. This probably represented the first appearance of FcR+ B lymphocytes even though cells bearing FcR were detected in fetal liver of all ages (days 12 to 18). The second period was around 10 days after birth when the proportion of Ig+ cells again increased concomitant with the appearance of CR+ nonadherent cells.     

Origin of the central cells of erythroblastic islands in fetal mouse liver: ultrahistochemical studies of membrane-bound glycoconjugates

 To clarify the origin of the central cells in hepatic erythroblastic islands, glycoconjugates on the surface of cellular constituents in fetal mice liver were ultrahistochemically examined using lectin staining. At 11 days of gestation, the cells derived from mesenchyme in fetal liver, including sinusoidal macrophages, endothelial cells, and erythropoietic cells, bound Griffonia simplicifolia isoagglutinin I-B4 (GS-I-B4), but hepatocytes lacked binding sites for the isolectin. Scavenger macrophages in the hepatic cords at 13 days of gestation and the central cells in the erythroblastic islands at 15 days of gestation also bound GS-I-B4. Hepatocytes, however, exhibited no GS-I-B4 binding site at any gestational day. At 11 days of gestation, none of the cells in fetal liver had binding sites for soybean agglutinin (SBA), but cells derived from mesenchyme acquired these binding sites at 13 days of gestation. The central cells in the erythroblastic islands also bound SBA, but hepatocytes did not bind the lectin at all. The central cells in the erythroblastic islands can be considered to belong to a mesenchymal cell lineage, and primitive sinusoidal macrophages at 11 days of gestation are possible precursors of these central cells. Accepted: 22 January 1997     

Partial characterization of Ia antigens from murine lymphoid cells

Congenic anti-Ia antisera were used to bind radiolabelled Ia antigens from cells of various strains of mice of knownH-2 haplotype. The results indicate that Ia antigens are proteins of molecular weight 30,000 to 35,000 daltons. The Ia antigens are distinct from known H-2 antigens as judged by independent immunoprecipitation as well as by molecular weight. Ia antigens are synthesized by, and are present on the surface of lymphoid cells as evidenced by incorporation studies using³H-leucine and enzymatic radioiodination of cells, respectively. Tissue distribution of cell surface Ia suggests that Ia antigens are on B cells. Ia antigens were detected in the incubation media of³H-leucine labeled splenocytes suggesting that antigens may be secreted.     

The appearance of surface H-2 antigens on fetal and postnatal murine hepatocytes in vivo and in vitro: A comparative study of MHC control

We have collected data on the kinetics of in vivo development of H-2^b antigens in fetal and postnatal hepatocytes from days 15–89 postcoitum (pc) in C57BL/10 mice using a sensitive immunoferritin labeling method combined with electron microscopy. We compared these data with data on the kinetic development of H-2^b antigens on fetal hepatocytes cultured from days 15, 16, and 17 pc onwards. Using the same techniques, we also compared the surface concentrations of H-2 antigens on the hepatocytes of pregnant and age-matched male and virgin female mice at day 110 pc. We found that the surface concentration of H-2 antigens on fetal and postnatal hepatocytes increased with time but that the birth event was associated with an increase in H-2 antigen expression from 60–80 ferritin grains per meter (F/m) to 190 F/m followed by a decrease to 115 F/m within 72 h of birth. The concentration of antigens on postnatal hepatocytes increased gradually and reached a maximum of 640 F/m on day 41 pc. By day 89 pc, however, this level had decreased 433 F/m, which was not significantly different from that found in day 110 pc males and virgin females. In contrast to previous findings, we found that hepatocytes cultured from days 15, 16, and 17 pc exhibited a large increase in H-2 surface antigen concentration within 48 h from 60–80 F/m to 1500–2200 F/m. After day 2, the H-2 concentration decreased and by days 14–17 in culture it was not significantly different from day 110 pc male and virgin female levels (485 F/m). Lastly, we found that the H-2 concentration on hepatocytes from pregnant mice was increased significantly (725 F/m) compared with the concentration of hepatocytes from day 110 pc males or virgin females. We postulate that the control of cell-surface major histocompatibility complex antigen concentration is achieved by a combination of intra and extracellular mechanisms and we discuss how this might be of benefit to the animal in vivo.     

Characterization of a spontaneous murine B cell leukemia (BCL1). I. Cell surface expression of IgM, IgD, Ia, and FcR.

The surface marker expression of a spontaneous B lymphocyte leukemia discovered in a BALB/c mouse (BCL1) was examined and found to include a subset of markers known to occur on normal B lymphocytes. The tumor cells bore surface Ig that included both mu- and delta-chains associated with the lambda light chain. Alloantigens coded for within the murine MHC, including H-2D, H-2K, and I-region products, were identified on the tumor cells. Although normal B lymphocytes are thought to express products coded for within both the I-A and I-E subregions, the BCL1 expressed only normal amounts of I-E subregion products. In addition, the H-2 and Ia antigens revealed by 2-dimensional gel electrophoresis exhibited an abnormal pattern of post-translational modifications. The Fc, but not the complement-receptor, was present on the surface of tumor cells. The presence of IgD, Ia antigens, and the responsiveness to lipopolysaccharide (see subsequent paper) have led us to postulate that the BCL1 tumor represents a later differentiative stage than murine B lymphocyte tumors previously described.     

Pyruvate kinase isoenzyme transitions in cultures of fetal rat hepatocytes

Changes in the expression of two isoenzymic forms of pyruvate kinase in fetal hepatocyte cultures derived from 15- and 19-day gestation rats are studied by immunocytochemical localization of the respective antigens. Initially, in cultures established from 15-day gestation rats only the ‘embryonic’ form of the enzyme (M2-PK) is detected in all cells. Cells which stain positively for the liver specific form of the enzyme (L-PK) are not observed. After 2 days' culture, a significant number of cells have become positive for L-PK. All the positive cells have a morphology which is typical of liver parenchymal cells. However, the majority of parenchymal cells remain negative for L-PK while retaining M2-PK. In contrast, all cells which display a fibroblastic morphology, as well as clear epithelial cells are M2-PK positive, but L-PK negative. In 5-day-old cultures, all hepatocytes have become L-PK positive. Hepatocytes derived from 19-day gestation rat liver stain positively for L-PK on day 1 of culture in agreement with previously published biochemical data. A minor population of negative cells is non-parenchymal in appearance. All parenchymal cells are negative when the culture is stained with M2-PK specific antibody. Five days after the culture is established, many non-parenchymal cells are present. Such cells are L-PK negative and M2-PK positive and their presence in cultures derived from both 15- and 19-day gestation rats explains the persistence of M2-PK. This study reveals that during enzymic differentiation of fetal hepatocytes, all immature hepatocytes are initially capable of expressing M2-PK while they do not produce L-PK. During culture, a sub-population of these cells initiates synthesis of L-PK, indicating that only a fraction of the cells differentiate. At the same time, hepatocytes which do not stain for M2-PK appear, which suggests that cells which initiate L-PK synthesis have ceased to make M2-PK. Eventually all hepatocytes are L-PK positive and M2-PK negative, indicating that a switchover in expression of the pyruvate kinase isoenzymes has occurred.     

Isolation and characterization of murine Ia antigens

The isolation and characterization of Ia antigens from both lymphoid and nonlymphoid cells was attempted by SDS-polyacrylamide gel electrophoresis of radiolabeled, NP-40 solubilized, and anti-Ia precipitated lysates. The profiles obtained indicate that membrane proteins with a molecular weight of approximately 30,000 can be isolated from peripheral B but not from peripheral T cells. Ia antigens cannot be immunoprecipitated from cortisone-resistant thymocytes, total thymocytes, allogeneically activated T cells, Con A stimulated T cells, and anti-Ig immunoadsorbent purified T cells. Ia antigens seem to comprise only 1%–2% of labeled splenic intracellular and membrane-associated proteins. They differ from H-2 antigens and immunoglobulin H and L chains with respect to size and serological reactivity. Ia antigens cannot be found to be secreted from lymph node cells or splenocytes into the extracellular incubation media. Tissue distribution studies indicate that Ia antigens are present on macrophages, fetal liver cells, epidermal cells, and bone marrow cells. They have not been found on such tumor cells as myelomas, teratomas, and lymphocytic leukemias.     

Immunocytochemical localization of albumin,transferrin, angiotensinogen and kininogens during the initial stages of the rat liver differentiation

Summary Rat albumin, transferrin, angiotensinogen, T kininogen (TKg) and high molecular weight kininogen (HKg) gene expression was examined immunocytochemically in embryonic and fetal livers. All these plasmatic proteins, angiotensinogen excepted, are detected as early as day 11 of gestation in intestine epithelial cells and embryonic hepatocytes. Angiotensinogen becomes expressible only at day 13 of gestation. During the early fetal period, the protein immunostaining increases strikingly in parallel with the hepatocyte differentiation. Albumin and transferrin are highly expressed comparatively to kininogens and angiotensinogen. For the first time, specific HKg is demonstrated in the rat liver.     

Immunocytochemical localization of albumin, transferrin, angiotensinogen and kininogens during the initial stages of the rat liver differentiation

Rat albumin, transferrin, angiotensinogen, T kininogen (TKg) and high molecular weight kininogen (HKg) gene expression was examined immunocytochemically in embryonic and fetal livers. All these plasmatic proteins, angiotensinogen excepted, are detected as early as day 11 of gestation in intestine epithelial cells and embryonic hepatocytes. Angiotensinogen becomes expressible only at day 13 of gestation. During the early fetal period, the protein immunostaining increases strikingly in parallel with the hepatocyte differentiation. Albumin and transferrin are highly expressed comparatively to kininogens and angiotensinogen. For the first time, specific HKg is demonstrated in the rat liver.     

Hypophyseal corticotropic activity during perinatal period: ontogenesis and regulation

1. In the fetal rat hypophysis, the "opio-melano-corticotropic" cells were immunocytologically revealed earlier in the pars distalis (day 16) than in the pars intermedia (day 17). At an early stage of embryonic development, some factors, possibly of nervous origin, could exert inductive influence on the differentiation and/or the increase in the number of immunoreactive cells. 2. Several forms of ACTH, characterized by their apparent molecular weight, bioactivity and immunoreactivity, can be observed when acid extracts are subjected to gel filtration on Sephadex G 50 fine columns. The ratios between these ACTH forms change during fetal development; the rise in bioactive ACTH content during the last days of gestation, is correlated with an increase of the "intermediate" and "little" molecular forms of ACTH and a parallel decrease of the "big" form. 3. The hypophyseal corticotropic activity is highest on days 18-19 of gestation. Between days 17 and 19, the cortico-stimulating activity is largely independent of the hypothalamus although the fetal hypothalamus shows light hypophyso-stimulating influence. In contrast, between days 19 and 21, the hypophyseal corticotropic activity is mostly under the hypothalamic control; the ACTH release is greatly reduced in its absence. 4. Circulating corticosteroids exert a negative feedback directly at the hypothalamic and pituitary levels. Such negative feedback could explain the decrease of the corticotropic activity of the pituitary during the last three days of gestation and the first postnatal days. 5. The fetus at the end of gestation, as the newborn, is responsive to stress; however, the extent of the pituitary response is weaker than in the adult.     

Characterization of T-cell-soluble factors modulating the expression of Ia and H-2 antigens on BALB/c B lymphoma cell lines

The effect of supernatants of concanavalin A-activated spleen cells (CAS) on the expression of various antigens, especially Ia antigens, on BALB/c B lymphoid cells, was examined. This study demonstrates the following: (i) CAS enhanced the expression of Ia antigens on four out of five BALB/c lymphoid cell lines. (ii) CAS selectively modulates the expression of Ia and H-2D, but not sIgM or viral gp70 expression, on X16C 8.5 tumor cells. The enhanced levels of Ia expression on B lymphoid tumor cells were also detected by using anti-Ia monoclonal antibodies. (iii) The molecular weight of soluble factor(s) affecting Ia and H-2 was approximately 40,000 estimated by gel filtration on a Sephadex G-200 column. (iv) Type 1 interferon but not interleukin 1, interleukin 2, or T-cell-replacing factor enhanced the expressions of Ia and H-2D antigens. (v) The activity of CAS-modulating Ia and H-2 antigens was eliminated by acidic treatment. It was concluded from this study that at least one of the factor(s) in CAS, modulating the antigenic expression of B-lymphoid cells, was interferon-like in nature. From our findings, a possible immunoregulatory mechanism by interferon was suggested: T cells, after stimulation of mitogens or antigens, secrete interferons which modulate the expression of Ia and H-2 on B cells. Then B cells, whose Ia and H-2 were modulated selectively by T-soluble factors(s), might interact with T cells much more efficiently.     

Affinity-dependent alterations of mouse B cell development by noninherited maternal antigen

We have examined the passage of maternal cells into the fetus during the gestation and postpartum in mice. Using enhanced green fluorescent protein (EGFP)-transgenic females, we showed that maternal cells frequently gain access to the fetus, mostly in syngeneic pregnancies, but also in allogeneic and outbred crosses. EGFP-transgenic cells, including B, T, and natural killer cells, can persist until adulthood, primarily in bone marrow and thymus. We then asked whether maternal cells, bearing antigens not inherited by the fetus, influence the development of fetal and neonatal B lymphocytes. We have used the B cell receptor 3-83 mu/delta transgenic mouse model, whose B cells recognize the major histocompatibility complex class I molecules H-2Kk and H-2Kb, with a high or moderate affinity, respectively. The fate of transgenic B cells in animals exposed to noninherited H-2Kk or H-2Kb maternal antigens (NIMA) during gestation and lactation was compared with those of nonexposed controls. In H-2Kk-exposed fetuses, NIMA-specific transgenic B cells are partially deleted during late gestation. Nondeleted cells have downmodulated their B cell receptor. In contrast, in NIMA H-2Kb-exposed neonates, transgenic B cells present an activated phenotype, including proliferation, upregulation of surface CD69, and preferential localization in the T cell zone of splenic follicles. This state of activation is still clearly detectable up to 3 wk of age. Thus, we show that fetal and neonatal B cell development is affected by maternal cells bearing antigens noninherited by the fetus and that this phenomenon is highly dependent on the affinity of the B cell receptor for the NIMA.     

Appearance of functional EGF receptor kinase during rodent embryogenesis.

Mouse and rat embryonic tissues at various stages of development were examined for epidermal growth factor (EGF) receptor kinase activity. The phosphorylated EGF receptor from embryonic tissues appeared as a band of mol. wt. 170 000 daltons on SDS gels. It was clearly demonstrable in the developing mouse fetus from 10 days of gestation onwards. The distribution of the EGF receptor kinase was studied in various tissues of 13 day mouse fetuses. The activity was apparent in the skin, developing skeletal muscles and various internal organs but was notably absent in the liver and brain. The amnion was found to be one of the richest sources of activity while the yolk sac was negative, and the placenta was weakly positive. In 16 day rat fetuses the distribution was quite similar to that of the 13 day mouse fetus. The liver acquired EGF receptor kinase activity by 18 days of gestation and had high activity in neonates. Phosphoamino acid analysis revealed that phosphotyrosine was the major labelled amino acid residue in the embryonic tissues. Thus, the EGF receptor of fetal tissues as studied by immune precipitation and phosphorylation appears to be a similar entity to that found in adult mammalian tissues. This functional EGF receptor kinase activity could first be detected at the time of onset of organogenesis.     

Soluble factors produced during an immune response regulate Ia antigen expression by murine adenocarcinoma and fibrosarcoma cells

LT-85 is an alveologenic adenocarcinoma of C3Hf/HeN mice. Comparisons of the in vitro and in vivo surface properties of these cells revealed that under normal conditions, they expressed I-A and I-E antigens iv vivo only. By using clonally derived cells, it was established that this phenomenon was not due to the selection of an Ia antigen-positive tumor cell subpopulation, but resulted from phenotypic conversion of Ia antigen-negative tumor cells. These tumor cells and 1053 cells (a fibrosarcoma of C3H/HeN MTV- mice) could, however, be induced to express I-A, I-E, and much higher levels of H-2 antigens in vitro by co-culturing them with spleen cells from LT-85 tumor-bearing C3H/HeN MTV- mice. In vitro induction of Ia and H-2 antigens did not result from contaminating splenocytes or from antigen transfer, because splenocytes from BALB/c (H-2d) mice immunized with A/J (H-2k/d) cells were able to induce the expression of Iak antigens by both tumor cell lines. It was found that this phenomenon was neither H-2-restricted nor antigen-specific. The results clearly indicated, however, that an immune response was required to generate phenotypic conversion of the tumor cells, both in vivo and in vitro. It was further found that soluble, rather than cellular, factors produced during an immune response induced the expression of Ia antigens by LT-85 and 1053 tumor cells. In contrast to what has been reported about the induction of Ia antigens on macrophages and normal epithelial and endothelial cells, the induction of Ia antigens on LT-85 and 1053 cells did not appear to require T cells, and did not involve gamma-interferon. These findings demonstrate that some tumor cells are capable of altering their MHC antigen phenotype in response to factors produced during an immune response in vivo or in vitro. Because of the involvement of Ia antigens in several aspects of immune phenomena, the ability of tumor cells to differentially express Ia antigens in response to environmental factors may have profound effects on host-tumor interactions. Furthermore, the differences seen in the phenotypes of tumor cells grown in vitro and in vivo suggest that in vitro methodologies of tumor cell characterization may not present a complete picture of the natural state of the tumor cell surface.     

Common antigen of oval and biliary epithelial cells (A6) is a differentiation marker of epithelial and erythroid cell lineages in early development of the mouse

Abstract. The A6 antigen - a surface-exposed component shared by mouse oval and biliary epithelial cells - was examined during prenatal development of mouse in order to elucidate its relation to liver progenitor cells. Immunohistochemical demonstration of the antigen was performed at the light and electron microscopy level beginning from the 9.5 day of gestation (26–28 somite pairs).
Up to the 11.5 day of gestation A6 antigen is found only in the visceral endoderm of yolk sac and gut epithelium, while liver diverticulum and liver are A6-negative. In the liver epithelial lineages A6 antigen behaves as a strong and reliable marker of biliary epithelial cells where it is found beginning from their emergence on the 15th day of gestation. It was not revealed in immature hepato-cytes beginning from the 16th day of gestation. However weak expression of the antigen was observed in hepato-blasts on 12–15 days of gestation possibly reflecting their ability to differentiate along either hepatocyte or biliary epithelial cell lineages.
Surprisingly, A6 antigen turned out to be a peculiar marker of the crythroid lineage: in mouse fetuses it distinguished A6 positive liver and spleen erythroblasts from A6 negative early hemopoietic cells of yolk sac origin. Moreover in the liver, A6 antigen probably distinguishes two waves of erythropoiesis: it is found on the erythroblasts from the 11.5 day of gestation onward while first extravascular erythroblasts appear in the liver on the 10th day of gestation. Both fetal and adult erythrocytes are A6-negative.
In the process of organogenesis A6 antigen was revealed in various mouse fetal organs. Usually it was found on plasma membranes of mucosal or ductular epithelial cells. Investigation of A6 antigen's physiological function would probably explain such specific localization.     

Studies of in vitro activation and differentiation of human B lymphocytes. I. Phenotypic and functional characterization of the B cell population responding to anti-Ig antibody

Investigation of the activation of splenic B cells by anti-immunoglobulin (Ig) antibody has enabled us to characterize the anti-Ig-responsive B cell and to analyze the phenotypic changes which accompany proliferation and differentiation. The anti-Ig antibody-responsive B cell population was characterized by the expression of high levels of the B2 antigen and represented approximately 40% of splenic B cells. Brisk mitogenesis which peaked at 3 to 4 days was induced by anti-Ig antibody. The proliferative phase was characterized phenotypically by a dramatic decline in B2 antigen expression, with most cells showing no detectable B2 by 4 days post-activation. The other hallmark of this phase was de novo expression of a group of "activation antigens." These included the B cell-restricted antigens B-LAST 1, BB1, and B5, and the T cell-associated interleukin 2 receptor and T12 antigens. Concomitantly, B1, B4, and Ia expression increased, the increase being roughly proportional to the increase in cell size. After day 4, the mitogenic response progressively diminished, while Ig synthesis increased. During this differentiation phase, cell surface antigens again displayed a distinct sequence of changes. The five activation antigens and the B1, B4, and Ia antigens began to decrease. However, two markers, T10 and PCA-1, which are found on plasmacytomas, appeared and their level of expression steadily increased. These changes and the appearance of morphologically identifiable plasma cells required the presence of T cells in this system. T cell supernatants alone induced Ig secretion but did not induce expression of PCA-1 or the appearance of cells with plasma cell morphology. The culture system developed in this study has allowed us to analyze the antigenic changes following activation by anti-Ig antibody. This sequence of changes has not only permitted the identification of antigens which, by their appearance at distinct stages may have an important role in proliferation and differentiation of B cells, but also provides us with the means of studying the function of each antigen.     

Isolation of twelve monoclonal antibodies against Ia and H-2 antigens. Serological characterization and reactivity with B and T lymphocytes

The cell hybridization technique was used for the production of 12 monoclonal antibodies against H-2K^k, H-2D^b, I-A^k and I-E^k antigens. The strain distribution pattern indicated that three antibodies reacted with new H-2 and Ia determinants, respectively, while the majority of determinants defined by the monoclonal antibodies showed good correlation with H-2 and Ia determinants described by conventional alloantisera.Monoclonal Ia antibodies showed strong reactivity with about 90% of surface IgM positive B cells, but not with T cells. In double fluorescence studies, both I-A and I-E determinants were always found to be coexpressed on the same B cells. When the high sensitivity of the fluorescence activated cell sorter was utilized, about 30 to 40% of purified lymph node T cells were found to carry both I-A and I-E antigens, although in a much lower density than B cells. In conclusion, monoclonal Ia antibodies appear to display the same serological and cellular reactivity pattern as do conventional antisera.     

The distribution of Ia antigens on the surfaces of lymphocytes.

The distribution of Ia antigens was studied on murine spleen lymphocytes by an ultrastructural technique employing deep freeze-etched replicas. Ia antigens were labeled on cells from appropriate congenic and recombinant strains of mice by incubating the cells with FITC-conjugated anti-Iak antibody, followed by ferritin-coupled Fab anti-FITC. Ia antigens were detected predominantly on immunoglobulin (Ig)-bearing B lymphocytes. Antigens coded for by the entire Ik region were present on the surfaces of 95% of the positive cells (from B10.BR mice) in densely packed microclusters. Ia specificities coded for by the I-A and I-C subregions (on 4R and B10.HTT mice) exhibited a more variable pattern, with 30 to 35% of the labeled cells having sparsely distributed Ia antigens in relatively discrete microclusters. Binding of anti-Iak antibody at 37 degrees C led to patch formation but not to capping. Modulation of surface Ig left Ia antigens diffusely distributed on the cell surface, indicating that these two membrane proteins are independent molecules.     

Temporal Characteristics of the Differentiation of Embryonic Erythroid Cells in Fetal Peripheral Blood of the Syrian Hamster

The morphological changes in erythroid cells and their nuclei in the circulation of fetuses of the Syrian hamster were investigated by use of an image-processing system. The analysis included monitoring of nuclear condensation, nuclear periphralization (access of the nucleus to the cell membrane), enucleation, density of cells, and changes in cell size from day 9 of gestation to day 5 after birth. The yolk-sac-derived erythroid cells made rapid progress in nuclear condensation on day 11, while this process proceeded at a much lower rate after day 12 of gestation. The peripheralization of nuclei started on day 10 and reached a maximum on day 11. The frequency of enucleated cells was below 2% on day 11, while it increased to 30% on day 12. Extruded nuclei, most of which were accompanied by a small quantity of cytoplasm, appeared in the circulation on day 12. The most frequently observed diameter of enucleated erythrocytes, which was 10–10.5 μm on day 12, fell gradually to 8–9 μm on day 14. By contrast, the shift from fetal liver erythrocytes to adult erythrocytes occurred in a discontinuous manner. Adult-type erythrocytes were detected after birth with diameters of 5.5–6 μm. Our data allows us to present the schedule of morphological changes during embryonic erythropoiesis and show that the developmental behavior of "primitive" yolk-sac-derived erythroid cells is more closely correlated with that of the "definitive" fetal liver cells than has been considered to be the case to date.