Mouse Yolk Sac Research Articles

Expression of the two adult β-globin genes in mouse yolk sac and fetal liver erythrocytes

The two adult β-globin genes (β1 s2major and β2 sminor) of the Hbb s2 haplotype are differentially expressed during development. Centrifugal elutriation was used to separate the two populations of erythrocytes present in developing fetuses. Hemoglobin analysis showed that the larger, nucleated erythrocytes (yolk sac-derived) have relatively larger amounts of β-sminor hemoglobin than do smaller, nonnucleated cells (fetal liver-, spleen-, and bone marrow-derived) at the same stage of development.

Development of thy 1-positive cells in mouse yolk sac cultured from 10 days of gestation

Development of thy 1-positive cells in mouse yolk sac cultured from 10 days of gestation

Vitamin D-dependent calcium-binding protein of mouse yolk sac. Biochemical and immunochemical properties and responses to 1,25-dihydroxycholecalciferol.

The present studies were performed to further characterize a mouse yolk sac protein which is similar or identical to the vitamin D-dependent intestinal calcium-binding protein (CaBP). Yolk sac protein and purified rat intestinal CaBP displayed full identity upon immunodiffusion (Ouchterlony) using antiserum to the rat intestinal CaBP. Immunoreactive CaBP in yolk sac homogenates eluted from gel permeation columns with the low molecular weight peak of 45Ca2+ binding (Chelex assay), and the electrophoretic mobility of the protein was markedly increased by EDTA. On days 11-13 of gestation, the concentrations of immunoreactive CaBP in yolk sac were 4-5-fold higher than in placenta; by days 16-17, the concentrations in yolk sac and placenta were similar. Incubation of yolk sac with [3H]leucine demonstrated synthesis of immunoprecipitable [3H]CaBP. A single band of 3H-labeled protein was seen on sodium dodecyl sulfate gel electrophoresis of the immunoprecipitate. This protein co-migrated with radioactive placental CaBP with an apparent Mr of 10,050. Addition of 1,25-dihydroxycholecalciferol (calcitriol) to organ culture media with or without serum increased the amount and concentration of CaBP in yolk sac (p less than 0.001) at 48 h. CaBP synthesis in yolk sac appeared to be independent of calcitriol concentrations in the maternal circulation since injection of the hormone into the maternal compartment produced no change in yolk sac CaBP despite increases of maternal intestinal and renal CaBP. These studies demonstrate that yolk sac immunoreactive CaBP is synthesized in yolk sac and has an apparent molecular size and calcium-binding properties characteristic of mammalian vitamin D-dependent calcium-binding proteins. The in vitro response of yolk sac CaBP to calcitriol is the first evidence of a vitamin D effect on the fetal membranes and suggests one function for calcitriol receptors in these tissues.

Xce genotype has no impact on the effect of imprinting on X-chromosome expression in the mouse yolk sac endoderm.

SummaryThe effect of theXce(x-chromosome controlling element) genotype on the randomness ofX-chromosome inactivation in the mouse was studied by monitoring the expression of theX-linked locuspgk-1. The main aim was to test whether theXcegenotype modified the preferential expression of the maternally derivedX-chromosome in the yolk sac endoderm. Quantitative electrophoresis of phosphoglycerate kinase (PGK-1) was used to studyPgk-1expression in the foetus, yolk sac mesoderm and yolk sac endoderm at 13½ dayspost coitum. TheXcea/Xcecgenotype caused non-randomX-chromosome expression in the foetus and yolk sac mesoderm. However, there was no evidence that theXcegenotype moderates the preferential expression of the maternally derivedX-chromosome in the yolk sac endoderm, as reported by Rastan & Cattanach (1983).

Immunochemical localization of vitamin D-dependent calcium-binding protein in mouse placenta and yolk sac.

A 10,000-dalton calcium-binding protein (10-kd CaBP) has been described in the placentae and yolk sacs of rats and mice. This protein is similar or identical to vitamin D-dependent intestinal CaBP and these proteins have been implicated in the molecular mechanisms of intestinal calcium absorption and transplacental calcium transport. Using an antiserum to the purified 10-kd rat intestinal CaBP, the localization of CaBP in the 16-17-day mouse yolk sac and placenta was studied immunocytochemically with peroxidase-antiperoxidase labelling and quantified by radial immunodiffusion. A high concentration of immunolabelling was observed in the endodermal cells of the intraplacental yolk sac lining the sinuses of Duval. The columnar endodermal cells lining one side of the endodermal sinuses adjacent to fetal vessels contained most of the immunoreactive label. Quantitation by radial immunodiffusion demonstrated a 5.5-fold higher concentration of CaBP in the portion of the placenta containing most of the intraplacental yolk sac than in the maternal half of the placenta. This demonstration of a 10-kd CaBP within the intraplacental yolk sac suggests this protein functions to facilitate placental calcium transport and is the first study which directly supports the hypothesis of a functional role for the sinuses of Duval in calcium transport.

Detection of macrophages and the characterization of Fc receptor-bearing cells in the mouse decidua, placenta and yolk sac using the macrophage-specific monoclonal antibody F4/80

A mouse macrophage-specific rat monoclonal antibody, F4/80, has been used to detect directly macrophages in short term cultures of mouse decidua, fetal placenta and yolk sac and to investigate the identity of Fc receptor (FcR) bearing cells in these tissues. We find that a significant proportion of FcR positive cells in decidual, placental and yolk sac tissues are macrophages as defined by the expression of the macrophage marker, F4/80 antigen. Macrophages may act as immunocompetent cells near to the maternal-fetal interface and play a significant role in the mechanism of the transfer of passive immunity from mother to fetus across the mouse yolk sac.

Selective expression of adult β-globin but not embryonic y-globin genes in hybrids of mouse yolk-sac and erythroleukemia cells

Hybrid cells from mouse erythroleukemia and yolk-sac cells from 12-day-old mouse embryos were formed, and the expression of adult β-globin and embryonic y-globin genes was examined. In these hybrid cells, hemoglobin synthesis could be induced by the addition of dimethylsulfoxide. The adult β-globin genes from both parental chromosomes were expressed, but embryonic y-globin genes were not. These results suggest that the expression of adult genes dominates that of embryonic genes.

Mouse yolk sac and intraembryonic tissues produce factors able to elicit differentiation of erythroid burst-forming units and colony-forming units, respectively.

This work was aimed at elucidating the environmental conditions that account for the production of embryonic erythrocytes in the mouse yolk sac (YS), while the adult-type hemoglobin and erythrocytes are generated in the fetal liver. Differentiation of YS hemopoietic stem cells (YS-HSC) of 9.5-day mouse embryos (prior to the colonization of the liver rudiment by HSC) was investigated in vitro. The influence of well-characterized erythroid growth factors, burst-promoting activity (BPA) and erythropoietin (EPO), which trigger the differentiation of early erythroid burst-forming units (BFU-E) and late erythroid colony-forming units (CFU-E), respectively, was tested on the YS-HSC in two different systems of culture: (i) organ culture and (ii) clonal culture in methylcellulose. When studied in organ culture, where the YS microenvironment was maintained, YS-HSC required only additional EPO to attain complete maturation into adult erythrocytes within 7 days. In contrast, YS hemopoietic single cells grown in methylcellulose, and thus released from the influence of the YS, required the presence of both BPA and EPO to generate BFU-E-derived colonies synthesizing high concentrations of hemoglobin. It is concluded that 9.5-day YS from mouse embryos is by itself able to promote the first differentiation steps of the adult lineage YS-HSC due to an intrinsic production of a BPA-like activity. In contrast, these experiments demonstrate that EPO or an EPO-like activity is not produced by YS tissues. As demonstrated earlier, if embryonic tissue is added to YS organ culture, although separated from it by a filter preventing cell contact, YS-HSC differentiate into adult erythrocytes producing adult-type hemoglobins. This shows that, in contrast to YS tissues, the early embryo produces EPO or a factor that can substitute for EPO.

Differential expression of albumin and α-fetoprotein genes in fetal tissues of mouse and rat

We have carried out a comparative analysis of the expression of the albumin and α-fetoprotein (AFP) genes in yolk sac and liver at different stages of fetal and postnatal life, in rat and mouse. Albumin and AFP mRNA levels were examined in these tissues by R 0 t analysis of RNA excess-cDNA hybridization data and/or by Dot blot hybridization. In addition, size analysis of the mRNA sequences were performed by electrophoretic fractionation on agarose gels containing methylmercury hydroxide and hybridization to radioactive cloned rat and mouse albumin and AFP cDNA probes. In the mouse, substantial amounts of albumin mRNA molecules were found in the yolk sac at different stages of development, while minimal levels of albumin mRNA sequences were detected in the rat yolk sac. The mouse yolk sac albumin mRNA molecules were found to be associated with the polysomes and to be functional in cell-free translation systems. In the rat, a reciprocal relationship appears to exist between the concentrations of the two mRNAs in yolk sac and embryonic liver. In contrast, in the mouse a parallel increase in both albumin and AFP mRNA levels was found in these tissues during fetal development. These results suggest that the expression of the albumin and AFP genes may be subjected to different regulatory events in these two members of the Muridae family.

FcR bearing cells within the mouse placenta and yolk sac express the macrophage specific antigen F4/80

FcR bearing cells within the mouse placenta and yolk sac express the macrophage specific antigen F4/80

Secretion and glycosylation of alpha-foetoprotein by the mouse yolk sac.

Secretion and glycosylation of alpha-foetoprotein (AFP) by mouse yolk sac were studied by using yolk-sac explants cultured in vitro. Yolk-sac explants rapidly incorporated [35S]methionine into AFP, whereas radioactively labelled AFP was not found in the medium until 30 min after incubation was initiated. Electrophoretic analysis revealed that microheterogeneity of AFP synthesized in explants increased in parallel with the gestational age of the yolk sacs. The change in microheterogeneity was noted by the formation of increasingly acidic forms. Only the most acidic forms of AFP were found to be present in the medium on each gestational day studied. Tunicamycin reduced the incorporation of glucosamine into AFP with a concomitant decrease in molecular weight and microheterogeneity. However, the relative amount of AFP released into the medium was not altered by the presence of tunicamycin. The presence of under-glycosylated AFP in the medium indicates that glycosylation of AFP is not essential for its secretion from the yolk sac. In light of these and previous findings, it is suggested that the glycosylation of AFP may be important for the turnover of this glycoprotein in serum.

Presence of mast cell precursors in the yolk sac of mice

Concentration of mast-cell precursors in hematopoietic tissues of mouse embryos was evaluated by a limiting dilution method. Cells from yolk sacs, livers, and bodies of (WB × C57 BL/6)F 1 (hereafter called WBB6F 1)-+/+ embryos were injected directly into the skin of adult WBB6F 1- W W v mice which were genetically depleted of tissue mast cells. Concentration of mast-cell precursors was calculated from the proportion of injection sites at which mast cells did not appear. Since the concentration of mast-cell precursors in the yolk sac was about 30 times as great as that of embryonic body at Day 9.5 of the pregnancy, the mast-cell precursors seemed to be generated within the yolk sac. The concentration in the yolk sac reached the maximum level at Day 11, and then dropped markedly at Day 13. In contrast, mast-cell precursors increased from Day 11 to Day 15 in the fetal liver. As a result, the concentration of 11-day yolk sacs was comparable to that of 15-day fetal liver. Although intravenous injection of 15-day fetal liver cells (2 × 10 6) rescued the general mast-cell depletion of WBB6F 1- W W v mice, the intravenous injection of the same number of 11-day yolk sac cells did not rescue it. In contrast with fetal livers, yolk sacs scarcely contained hematopoietic stem cells which were measured by spleen colony formation. Therefore, the mast-cell precursors of the yolk sac may not originate from such stem cells.

Synthesis of secretory proteins in developing mouse yolk sac

Synthesis of secretory proteins in the developing mouse visceral yolk sac was studied. Newly synthesized proteins were labeled with [ 35S]methionine and characterized by two-dimensional gel electrophoresis. A large increase in the relative rate of synthesis of a small number of proteins occurred between Days 9.5 and 15.5 of development. These proteins were the predominant proteins synthesized and secreted by the yolk sac throughout this period of gestation. Two of these proteins were identified as α-fetoprotein and transferrin by specific immunoprecipitation. α-Fetoprotein synthesis increased from about 3% of the total protein synthesis at Day 9.5 to about 26% at Day 15.5 after which it declined slightly. The relative rate of transferrin synthesis had a similar developmental pattern, reaching the highest level (5%) at Day 15.5, but declined more rapidly than α-fetoprotein synthesis. Quantitatively, these two proteins represented about 60% of the total secreted protein. Gestational changes in the content of α-fetoprotein messenger RNA were determined by hybridization analysis using α-fetoprotein complementary DNA probe. The percentage of α-fetoprotein messenger RNA in total yolk sac RNA increased about ninefold from Day 9.5 to Day 14.5. This increase correlated well with the increase in the relative rate of α-fetoprotein synthesis during the identical period. This study suggests that after Day 9.5 the yolk sac is completing a differentiation process which is characterized by the preferential expression of a small group of secretory protein genes.

Stability of α-fetoprotein messenger RNA in mouse yolk sac

Incubation of mouse yolk sac explants in the presence of α-amanitin led to a 2.7-fold reduction in total protein synthesis and a 2-fold increase in the relative rate of α-fetoprotein synthesis. This indicated that α-fetoprotein messenger RNA was more stable than other yolk sac messenger RNA. This was further documented by pulse-chase experiments in which decay of α-fetoprotein messenger RNA was monitored by hybridization to filter-bound α-fetoprotein complementary DNA. α-Fetoprotein messenger RNA was found to be stable during a 30-hr-chase period whereas most other poly(A) + RNA species decayed with a half-life of 6 hr. There appeared to be no changes in the stability of α-fetoprotein messenger RNA from Day 11.5 to Day 17.5 of gestation. This supports the conclusion that the selective stabilization of α-fetoprotein messenger RNA is an important mechanism for maintaining the abundancy of this message in the developing yolk sac.

In vitro induction of adult erythropoiesis in early mouse yolk sac.

The capacity of yolk sac hemopoietic cells to produce either primitive or definitive erythrocytes was analyzed in vitro under three different experimental conditions. (i) Before the 28-somite stage (prior to colonization of the liver rudiment by hemopoietic cells), yolk sac explanted alone produced solely primitive erythrocytes and only for a short time. (ii) When allowed to colonize a liver rudiment, hemopoietic cells from the yolk sac gave rise to definitive erythrocytes. (iii) These cells could express the same capacity when stimulated by various intraembryonic organs, even if no direct cell--cell contact was established between stimulating tissue and target hemopoietic cells. These results provide evidence that humoral factors present in embryos past the 28-somite stage act on hemopoietic cells, inducing the onset of definitive erythropoiesis.

X-chromosome inactivation in extra-embryonic membranes of diploid parthenogenetic mouse embryos demonstrated by differential staining.

In somatic cells of female mammals one of the two X chromosomes is genetically inactive and heterochromatic, resulting in dosage compensation for X-linked genes. In marsupials the paternally derived X chromosome is preferentially inactivated. In eutherian mammals, although either X chromosome can be inactivated at random in somatic cells, preferential inactivation of the paternally derived X chromosome has been demonstrated cytologically in mouse and rat yolk sac and mouse chorion and biochemically in mouse yolk sac, chorionic ectoderm and trophoblast. In mouse yolk sac the non-random element has been shown both biochemically and cytologically to be confined to the endoderm layer in which there is almost total paternal X-chromosome activity in the separated yolk sac layers of diploid parthenogenetic mouse embryos in which both X chromosomes are maternally derived. Kaufman et al. have demonstrated X inactivation in somatic cells of diploid parthenogenetic embryos, and we have used a modification of Kanda's method, which renders the presumptive inactive X dark staining, to reveal an inactive X chromosome in both endoderm and mesoderm layers of separated yolk sacs from parthenogenones. Thus even in tissues in which there is normally total non-random paternal X inactivation, in the absence of a paternally derived X chromosome a maternally derived X can be inactivated.

Epithelium of mouse yolk sac placenta lacks H-2 complex alloantigens.

The only fetal cell membrane exposed to the mother in the mouse yolk sac placenta is the apical membrane of the endodermal epithelial cells. In yolk sac preparations in vitro, this apical membrane was exposed to reagents or cells in the incubation medium. By using several techniques we were not able to detect fetal major histocompatibility complex (MHC) antigens in this membrane. Immunoferritin labeling with and without prefixation and after neurominidase and trypsin digestion indicated that the apical membrane could contain no more than approximately 1% of the H-2 complex antigens that were present on peritoneal macrophages. Incubation of yolk sac preparations in anti-H-2 complex antiserum and complement had no cytotoxic effect on the endodermal epithelium, nor did incubation in an excess of alloreactive lymphocytes. Dissociated preparations of prefixed yolk sac contained endodermal epithelial cells and vascular endothelial cells whose entire surface membranes were exposed to the medium. H-2-complex antigens were not detected by immunoferritin labeling in either the apical or the laterobasal membrane of the yolk sac endoderm, but they were present in low density on the vascular endothelium. Also, incubation of unfixed, dissociated cells in anti-H-2-complex serum and complement had no detectable cytotoxic effect on endodermal epithelial cells. These observations indicate that H-2 antigens are sparse or absent in both the apical and laterobasal membranes of endodermal epithelial cells. The deficiency of MHC antigens in the apical membrane may account for the failure of sensitized females to reject the yolk sac, whereas the composition of the laterobasal membrane is probably less important to maternal-fetal relations. The present observations are consistent with labeling studies of adult-lining epithelial cells, which indicate that self-marker MHC molecules are absent from the apical membranes oriented toward the outside world and variably expressed in the laterobasal self-side membranes. It is suggested that the corresponding exclusion of fetal self-marker molecules from the apical membranes of some kinds of placental epithelia would deprive the mother of target sites for an alloimmune reaction at the maternal-fetal interface.

Studies on mouse autoreactive cells: III. Fetal development of autorosette-forming cells

Self-recognition assessed by rosette formation by lymphocytes with erythrocytes of syngeneic or autologous origin is a very primitive function that is present before lymphoid system development proper in the thymus. Autologous rosette-forming cells (A-RFC) have been found in the very early yolk sac of pregnant mice of 10–11 days gestation. Moreover, when these 10- to 11-days' gestation pregnant mice were subcutaneously injected with facteur thymique sérique (FTS) 1 day before A-RFC examination, it appears that FTS reduces the number of A-RFC in the yolk sac by 63%. Thus it has not been possible to determine whether FTS acted by changing the migration capacity or the expression of receptors on the cell surface.

Molecular cloning of DNA complementary to a mouse α-fetoprotein mRNA sequence

DNA complementary to mouse yolk sac messenger RNA has been inserted at the PstI site of the plasmid pBR322 by annealing of the oligo(dG)-tailed plasmid DNA with the oligo(dC)-tailed mouse DNA. Transformation of Escherichia coli strain RRI with this annealed DNA yielded clones bearing recombinant plasmids. The clones were screened for DNA complementary to mouse a-fetoprotein (AFP) messenger RNA sequences by hybridization with a cDNA probe transcribed from an AFP mRNA of over 90% purity. Out of nine plasmids that were isolated and analyzed by restriction mapping, all had homologous insert DNA of various lengths. The plasmid with the longest insert, pAF6, contained 1.65 kb of added DNA, which is about 70% of the AFP mRNA. This clone was positively identified by a hybridization-translation procedure to contain a cDNA sequence for AFP. A restriction map of this clone and the orientation of the message are presented.

Isolation of alpha-fetoprotein messenger RNA from mouse yolk sac.

Isolation of alpha-fetoprotein messenger RNA from mouse yolk sac.