Identification of mature plasma cells in early rat yolk sac. A possible origin from the endodermal cell layer: immunohistochemistry and immunoelectron microscopic study

Plasma cells play a pivotal role in the immune system and are responsible for the synthesis and release of immunoglobulins. Numerous in vitro culture experiments on the yolk sac demonstrated the generation of mature cells of the myeloid and lymphoid lineages under appropriate culture conditions. However, there are no reports describing the development of mature lymphoid cells in the yolk sac so far. For this reason, we undertook this study to investigate the development of antibody-containing plasma cells during early yolk sac haematopoiesis. Immunohistochemistry and immunoelectron microscopy were employed in the study. Results of this work demonstrated very weak immune staining for the intracytoplasmic IgA, IgG, and IgM at days 10 and 11 of embryonic life, while dark staining was obtained at 12 days. Positive staining was localized to the endodermal cell layer. Electron microscopic examinations revealed the existence of cells with the typical characteristics of plasma cells inside the endodermal cell layer, which may suggest their endodermal origin. To further verify the nature of these cells, intracytoplasmic immunoglobulins were demonstrated by immunoelectron microscopy. The present study demonstrated emergence of mature functioning plasma cells in early rat yolk sac. In a previous work we hypothesized the possibility of endodermal origin of yolk sac macrophages. This study adds additional evidence to support that hypothesis. The possible role of plasma cells in the yolk sac is discussed.     

Isolation and characterization of the Fc receptor from the fetal yolk sac of the rat

The yolk sac of the fetal rat and the proximal small intestine of the neonatal rat selectively transport maternal IgG. IgG-Fc receptors are thought to mediate transport across the epithelium of both tissues. We used a mouse mAb (MC-39) against the 45-54-kD component of the Fc receptor of the neonatal intestine to find an antigenically related protein that might function as an Fc receptor in fetal yolk sac. In immunoblots of yolk sac, MC-39 recognized a protein band with apparent molecular mass of 54-58 kD. MC-39 bound to the endoderm of yolk sac in immunofluorescence studies. In immunogold-labeling experiments MC-39 was associated mainly with small vesicles in the apical cytoplasm and in the region near the basolateral membrane of endodermal cells. The MC- 39 cross-reactive protein and beta 2-microglobulin, a component of the intestinal Fc receptor, were copurified from detergent-solubilized yolk sac by an affinity purification that selected for proteins which, like the intestinal receptor, bound to IgG at pH 6.0 and eluted at pH 8.0. In summary, the data suggest that we have isolated the Fc receptor of the yolk sac and that this receptor is structurally and functionally related to the Fc receptor of the neonatal intestine. An unexpected finding is that, unlike the intestinal receptor which binds maternal IgG on the apical cell surface, the yolk sac receptor appears to bind IgG only within apical compartments which we suggest represent the endosomal complex.     

Formation sites and distribution of osteoclast progenitor cells during the ontogeny of the mouse

The presence of osteoclast progenitor cells in embryonic, fetal, young growing, and adult murine tissues and organs was investigated in a coculture system with fetal metatarsal bones stripped of periosteum and not yet invaded by osteoclasts. Osteoclasts were found to originate from the early yolk sac and from every tissue tested in the fetus and young mouse. In the adult mouse they were formed only from tissues with a large mononuclear phagocyte population. No osteoclasts could be generated from the young embryo proper, prior to establishment of the vascular connection with the yolk sac. Progenitors of osteoclasts or their stem cells therefore do not develop from undifferentiated mesenchyme outside the yolk sac, but are distributed from the yolk sac to embryonic tissues and hematopoietic organs through the vascular circulation. The embryonic distribution of osteoclast progenitors coincides with the distribution of immature macrophages. Furthermore, they are present before the formation of monocytes in the fetus. The results also indicate that osteoclast precursor cells are not identical with mature, differentiated macrophages, but are cells with little capacity to phagocytose and therefore are, at the most immature progenitors of macrophages or cells of an early diverging lineage. In view of these results the derivation of osteoclasts is discussed.     

Of birds and mice: hematopoietic stem cell development

For many years it has been assumed that the ontogeny of the mammalian hematopoietic system involves sequential transfers of hematopoietic stem cells (HSCs) generated in the yolk sac blood islands, to successive hematopoietic organs as these become active in the embryo (fetal liver, thymus, spleen and eventually bone marrow). Very little was known about early events related to hematopoiesis that could take place during the 4.5 day gap separating the appearance of the yolk sac blood islands and the stage of a fully active fetal liver. Experiments performed in birds documented that the yolk sac only produce erythro-myeloid precursors that become extinct after the emergence of a second wave of intra-embryonic HSCs from the region neighbouring the dorsal aorta. The experimental approaches undertaken over the last ten years in the murine model, which are reviewed here, led to the conclusion that the rules governing avian hematopoietic development basically apply to higher vertebrates.     

Prealbumin gene expression during mouse development studied by in situ hybridization

Localization of prealbumin mRNA in tissues from mice at various stages of gestation was investigated using in situ hybridization procedures. Prealbumin mRNA was detected as early as the 10th day of gestation. It was specifically localized in endodermal cells of the visceral yolk sac, tela choroidea, and hepatocytes. In the adult mice, prealbumin mRNA was localized in the hepatocytes and choroid plexus epithelial cells. These observations indicate that synthesis of prealbumin mRNA is initiated in several different types of cells at early stages of fetal development.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

Embryonic fates for extraembryonic lineages: New perspectives

The prevailing view of the functions of the extraembryonic lineages of the mammalian embryo has been that they serve solely to support its intrauterine development. In recent years, a number of studies have suggested that the extraembryonic mesoderm and visceral endoderm in fact contribute cells to tissues of the developing animal. In this mini‐review, we discuss evidence that the yolk sac is an early source of hematopoietic stem and progenitor cells and that the cells of the visceral endoderm, once thought to be segregated solely to the yolk sac, constitute a subpopulation of cells within the developing gut tube and perhaps other endodermal structures. Fascinating questions remain to be addressed and are likely to establish a new paradigm for studying early mammalian development. Understanding the processes that give rise to stem cell populations in development may lead to advances in stem cell therapies and regenerative medicine. J. Cell. Biochem. 107: 586–591, 2009. © 2009 Wiley‐Liss, Inc.     

Cultured chick yolk sac epithelium: structure and IgG surface binding

The chick yolk sac endoderm transports maternal immunoglobulin G (IgG) from the yolk into the embryo during development, providing the newly hatched chick with passive immunity until it becomes immunocompetent. To study this transport process, chick yolk sac endodermal cells isolated from embryos of 6 to 18 days of incubation were grown in vitro on a collagen substrate. The cultured cells possessed a remarkable structural similarity to the in vivo tissue and reformed a polarized confluent epithelium with tight junctions and desmosomes joining the cells at their apical margins. In addition, the cells exhibited apical microvilli, numerous phagolysosomes in the cytoplasm and retained the expression of the yolk sac endoderm-specific enzyme marker, cysteine lyase. Importantly, the cultured cells retained the ability to specifically bind IgG as demonstrated by indirect immunofluorescence. Chicken IgG bound to the cultured cells at 4 degrees C in a diffuse pattern that clustered into a punctate pattern when a second antibody was used. Cultures from yolk sacs of day 6 through day 18 of development all demonstrated this immunofluorescent labeling for at least 14 days in culture. These results demonstrate that cultured yolk sac endoderm maintains its differentiated morphology and ability to bind IgG.     

The yolk sac in late embryonic development of the stick insect Carausius morosus (Br.)

Differentiation of the yolk sac was examined ultrastructurally and cytochemically in late embryonic development of the stick insect Carausius morosus. During migration along the yolk sac, endodermal cells form a discontinuous cell epithelium, leaving wide intercellular channels between neighbouring cell clusters. Within the same cell cluster, cells are all joined by septate junctions. In the proximity of the proctodeum region, intercellular channels are filled with numerous cell debris which are shown to derive from vitellophages undergoing cell lysis. Yolk sacs resolved by gel electrophoresis are shown to release a number of vitellin polypeptides into the culture medium. These are equivalent in molecular weight to those present in the vitellophage yolk granules This observation is consistent with the evidence that the basement lamina may act as a course physical filter, retaining particles larger than colloidal thorium dioxide and allowing free percolation of peroxidase. Differentiating endodermal cells form a microvillar striated border along the apical plasma membrane. A number of vesicular criptae were frequently seen in these differentiating endodermal cells. Electron dense granules released by endodermal cells are suggested to play a role in vitellophage lysis and vitellin release from the enclosed yolk granules.     

Hemopoiesis in the human yolk sac

The endodermal layer of the human yolk sac was examined three-dimensionally with light microscopy on serial sections using scanning electron microscopy and transmission electron microscopy to find the origin of hemopoiesis in the yolk sac. Cell-labelling techniques were also employed using the monoclonal anti-transferrin receptor antibody. Orifices of the endodermal and intracellular tubules facing the yolk-sac cavity were demonstrated on the endodermal surface. Various-sized blood cells in various stages of differentiation and maturation were distributed in the yolk-sac cavity and tubules and were observed also at the orifices of the tubules. The morphological and the immunological findings suggest that blood cells with large nuclei in the endodermal layer are the most immature. The present results suggest that blood cells originate from the endodermal layer and are carried to the embryo through the yolk sac cavity and the vitelline duct. It is probable that the endodermal and intracellular systems of tubules have an important role in the transport of blood cells, including stem cells.     

Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse.

In this study, we have mapped the onset of hematopoietic development in the mouse embryo using colony-forming progenitor assays and PCR-based gene expression analysis. With this approach, we demonstrate that commitment of embryonic cells to hematopoietic fates begins in proximal regions of the egg cylinder at the mid-primitive streak stage (E7.0) with the simultaneous appearance of primitive erythroid and macrophage progenitors. Development of these progenitors was associated with the expression of SCL/tal-1 and GATA-1, genes known to be involved in the development and maturation of the hematopoietic system. Kinetic analysis revealed the transient nature of the primitive erythroid lineage, as progenitors increased in number in the developing yolk sac until early somite-pair stages of development (E8.25) and then declined sharply to undetectable levels by 20 somite pairs (E9.0). Primitive erythroid progenitors were not detected in any other tissue at any stage of embryonic development. The early wave of primitive erythropoiesis was followed by the appearance of definitive erythroid progenitors (BFU-E) that were first detectable at 1-7 somite pairs (E8.25) exclusively within the yolk sac. The appearance of BFU-E was followed by the development of later stage definitive erythroid (CFU-E), mast cell and bipotential granulocyte/macrophage progenitors in the yolk sac. C-myb, a gene essential for definitive hematopoiesis, was expressed at low levels in the yolk sac just prior to and during the early development of these definitive erythroid progenitors. All hematopoietic activity was localized to the yolk sac until circulation was established (E8.5) at which time progenitors from all lineages were detected in the bloodstream and subsequently in the fetal liver following its development. This pattern of development suggests that definitive hematopoietic progenitors arise in the yolk sac, migrate through the bloodstream and seed the fetal liver to rapidly initiate the first phase of intraembryonic hematopoiesis. Together, these findings demonstrate that commitment to hematopoietic fates begins in early gastrulation, that the yolk sac is the only site of primitive erythropoiesis and that the yolk sac serves as the first source of definitive hematopoietic progenitors during embryonic development.     

Identity of cells containing apolipoprotein B messenger RNA, in 6- to 12-week postfertilization human embryos

Apolipoprotein B (Apo B) mRNA has been localized by in situ hybridization to various cell types in the liver, gut and yolk sack of the 6- to 12-week postfertilization human conceptus. In the fetal liver it is probable that the immature hepatocytes contain Apo B mRNA. In the yolk sack, the Apo B cDNA probe hybridizes mainly to the large endodermal cells and in the fetal gut the epithelium seems responsible for the majority of Apo B mRNA production. The fetal brain did not show any detectable hybridization to the Apo B probe. Unlike the situation seen in the adult, immunoprecipitation experiments demonstrated that only the B100 form of the protein was synthesized and secreted by the liver, gut and yolk sack at this early stage of human development.     

Expression of the 14 kDa and 16 kDa galactoside-binding lectins during differentiation of the chick yolk sac

The gastrulating chick embryo expresses two galactoside-binding lectins of 14 kDa and 16 kDa. These lectins are present in the area pellucida and area opaca, and in the latter are concentrated in the endoderm. Since the area opaca is the progenitor of the yolk sac, we studied the galactose-binding lectins during the development of this extraembryonic organ. In the yolk sac, lectin expression surges between 2 and 4 days, and thereafter remains constant throughout development. Using monoclonal antibodies (mAbs) specific to the 16 kDa yolk sac lectin, and a panel of polyclonal antibodies to the 14 kDa and 16 kDa lectins we studied lectin expression. The mAbs inhibit the hermagglutinating activity of extracts from chick yolk sac, embryonic pectoral muscle, and adult liver, but have no effect on the hemagglutinating activity of extracts from the adult intestine. Immunolocalization studies with the mAbs and polyclonal antibodies indicate that in the less differentiated endodermal cells of the area vitellina the 16 kDa lectin is present in discrete lectin-rich inclusions. In contrast, within the maturing endodermal epithelium of area vasculosa the 16 kDa lectin is present around the intracellular yolk platelets, and is associated with the cytoplasmic matrix. The 16 kDa lectin is also found at the apical cell surface of the yolk sac epithelium, in some regions closely associated with the plasma membrane. The 14 kDa lectin is distributed intracellularly surrounding the yolk platelets of the maturing yolk sac endoderm. The surge in expression of the 16 kDa lectin at the time of expansion of the area opaca suggests that it may be involved in the spreading of this area. Our findings also indicate that as the yolk sac endoderm differentiates into an epithelium intracellular lectin expression changes from predominantly organelle associated to cytoplasm associated. The association of both lectins with yolk suggest that the lectins may also be involved in the processing of intracellular and extracellular yolk proteins. These results, in con junction with previous findings indicating the presence of these lectins in the extracellular matrix (Didier et al., Histochemistry 100:485, 1993; Zalik et al., Intl J Dev Biol 38:55–68, 1994) indicate that these lectins play multiple roles in embryonic development.     

Primitive lymphohematopoietic precursor cell lines generated in culture from day 7 early-mid-primitive streak stage mouse embryo.

During mouse development, the first lymphohematopoietic precursor cells and myeloid or erythroid cell lineage-determined cells can be detected in the yolk sac at days 8-8.5 of gestation. The characteristics of the cells that give rise to these yolk sac primitive lymphohematopoietic cells and the molecular events controlling this process remain poorly defined. We show here that cell suspensions from day 7 early-mid-primitive streak stage embryo proper generated early immature PgP-1+ Joro 177+ Lin- hematopoietic cells and some Mac-1+ myeloid and TER 119+ erythroid cells after co-culture with the yolk sac-derived stromal cell line YS6 without addition of exogenous cytokines. Purified Lin- hematopoietic cells generated in these cultures did not express genes known to be transcribed at early stages of lymphoid, myeloid or erythroid cell differentiation and were able to give rise to T and B lymphocytes, myeloid cells and erythroid cells after appropriate further induction in vitro. Several cell lines were established in culture with a mixture of four cytokines from the PgP-1+ Joro 177+ Lin- cell population. The cell lines shared phenotypic and genotypic characteristics with the PgP-1+ Joro 177+ Lin- cell population generated in culture from day 7 embryo proper and they were able to reconstitute the lymphohematopoietic system of irradiated mice. Taken together these results support a model of lymphohematopoiesis in which cells from day 7 early-mid-primitive streak mouse embryo proper migrate and colonize the visceral yolk sac. There they generate primitive lymphohematopoietic precursor cells and the first erythroid and myeloid hematopoietic cells under the influence of yolk sac stromal cells like the YS6 cells described here.     

Kit ligand has a critical role in mouse yolk sac and aorta–gonad–mesonephros hematopoiesis

Few studies report on the in vivo requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta–gonad–mesonephros (AGM) niche. In‐depth analysis of loss‐of‐function and transgenic reporter mouse models show that Kitl‐deficient embryos harbor decreased numbers of yolk sac erythro‐myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence. This EMP defect causes a dramatic decrease in fetal liver erythroid cells prior to the onset of hematopoietic stem cell (HSC)‐derived erythropoiesis, and a reduction in tissue‐resident macrophages. Pre‐HSCs in the AGM require Kitl for survival and maturation, but not proliferation. Although Kitl is expressed widely in all embryonic hematopoietic niches, conditional deletion in endothelial cells recapitulates germline loss‐of‐function phenotypes in AGM and yolk sac, with phenotypic HSCs but not EMPs remaining dependent on endothelial Kitl upon migration to the fetal liver. In conclusion, our data establish Kitl as a critical regulator in the in vivoAGM and yolk sac endothelial niche.     

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.     

The origin of chicken hematopoietic colonies as assayed in semisolid agar.

This investigation was undertaken to determine whether primitive stem cells and/or fully differentiated macrophages were the source of in vitro colonies derived from hematopoietic tissues. The chicken colony-forming cell (CFC) present in uncultured yolk sac was a nonadherent, presumably undifferentiated cell. The efficiency of colony formation in this case was approximately 0.08%. In contrast to uncultured yolk sac, the CFC present in one-week old yolk sac cultures was evidently a macrophage. Yolk sac cultures, which consisted of greater than 99% macrophages, produced colonies with an efficiency of 1-5% while cultures derived from peritoneal macrophages produced colonies with an efficiency of 10%. Silica selectively destroyed macrophages and reduced the colony forming efficiency of cells derived from yolk sac cultures.     

The human foetal yolk sac

Summary Specimens of human foetal yolk sac from conceptuses of 8 and 10 weeks menstrual age were studied with the electron microscope. At 8 weeks columns of endodermal cells projected into the underlying mesenchyme. Several types of endodermal cell were identified; some contained much granular endoplasmic reticulum and abundant glycogen; others resembled the haemocytoblasts present in the mesenchyme and yet others contained membrane-bounded channels similar to those seen in megakaryocytes. It was suggested that the endoderm is the site of origin of the blood cells but that, while the platelets may be formed within the endoderm, the normal development of the red cells is conditional upon their early release into the mesenchyme and possibly the attainment of an intravascular position. Intravascular macrophages were identified and their role in determining the nature of the blood picture during the period of functional acitvity of the sac discussed. The morphology of the epithelium on the external surface of the sac was discussed in relation to the possibility of its playing a part in the exchange of materials between the yolk sac and the chorionic cavity.Supported in part by grant no. 5-T01-GM-00582-08 from the U.S. Public Health Service.     

Yolk sac failure in embryopathy due to hyperglycemia: ultrastructural analysis of yolk sac differentiation associated with embryopathy in rat conceptuses under hyperglycemic conditions

Diabetes mellitus in pregnancy is associated with an increased incidence of various congenital anomalies that occur during organogenesis. Because a well functioning yolk sac is crucial to embryonic growth and development during this period, we performed an ultrastructural study of the effects of excess glucose (total glucose 750 mg/dl, osmolality 305 mOsm/kg) on pregnancy day 10 (Witschi stage 13) rat conceptuses cultured for 48 hr in heat-inactivated male rat serum with and without added d- or l-glucose. Embryos exposed to excess d-glucose demonstrated decreased conceptus size (P less than 0.001), and gross malformations in a dose-related fashion. The visceral yolk sac capillaries and vitelline vessels of conceptuses in excess d-glucose were sparse, patchy, and nonuniformly located. Ultrastructurally, the visceral yolk sac endodermal cells had reduced numbers of rough endoplasmic reticulum, ribosomes, and mitochondria. These obvious defects in yolk sac structure suggest that hyperglycemia during organogenesis has a primary deleterious effect on yolk sac function with resultant embryopathy.     

The association between prion proteins and Aβ₁₋₄₂ oligomers in cytotoxicity and apoptosis

Microglia are cells from non-neuronal lineages that reside in the central nervous system. In zebrafish, early macrophages migrate from the yolk sac to the brain and retina at 26-30 hour post fertilization (hpf) and transform into microglia at 55-60 hpf. The migration of macrophages into the central nervous system requires signaling by macrophage colony stimulating factor-1 receptor (csf-1r), which is encoded by the gene fms. In this study, we show that the targeted knockdown of csf-1r with morpholino oligonucleotides delays migration of macrophages from the yolk sac to the retina, and this delay in macrophage migration results in microphthalmia, delay in cell cycle withdrawal among retinal progenitors and the absence of neuronal differentiation. When embryos were allowed to survive beyond the time when morpholino-dependent translation inhibition is lost, microglia re-occupy the retina and neuronal differentiation partially recovers. Our data demonstrate that microglia are required for normal retinal growth and neurogenesis. This study provides new insight into the neurogenic role of microglia during retinal development in zebrafish.