An in vitro morphological study of the mouse visceral yolk sac and possible yolk sac immunocyte precursors

Summary Mouse visceral yolk sac has been organ cultured from 9 days of gestation, a time prior to the thymus being lymphoid, until 12 days of gestation, a time after which the thymus is lymphoid. During the culture period the endodermal epithelial cells survived well, erythropoiesis diminished, endothelial-lined cavities formed in the mesodermal mass, and cells developed which have been classified as large, medium and small immunocyte precursors. The cytoplasm of the immunocyte precursors contains polysomes, spherical mitochondria, a few profiles of rough endoplasmic reticulum, occasional granules and a large Golgi complex. This study offers morphological support for the yolk sac origin of immunocyte precursors in the mouse which may seed the thymus and liver.Supported by NIH Grant AI 13486-01     

Pattern of serum protein gene expression in mouse visceral yolk sac and foetal liver.

We have shown by molecular hybridisation that the mRNAs for albumin, transferrin, apolipoprotein-A1, and alpha 1-antitrypsin are expressed at high levels in mouse visceral yolk sac. In contrast, the mRNAs for contrapsin (a plasma protease inhibitor) and the major urinary proteins (MUPs) are not detected in the visceral yolk sac at any stage of embryonic development. Contrapsin and MUP mRNAs both appear late in liver development. These differences in expression suggest that the visceral yolk sac is more similar to the foetal than adult mouse liver in its pattern of gene expression. However, the developmental time course of expression of these mRNAs is different between the foetal liver and the yolk sac. Evidence is also presented that the visceral yolk sac synthesises and secretes other apolipoproteins in addition to apolipoprotein-A1. These results suggest that the visceral yolk sac and foetal liver, two tissues with different embryological lineages, perform similar functions but are independently programmed for expression of the same set of serum protein genes.     

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 alpha-fetoprotein messenger RNA in mouse yolk sac

Changes in the activity of DNA polymerase-α and in subcellular distribution were studied during gastrulation of the sea urchin, Hemicentrotus pulcherrimus. Although the activity of DNA polymerase-α for each embryo was constant up to the blastula stage as reported previously, the enzyme activity increased during gastrulation by about twofold prior to an increase in its DNA content. Thereafter the enzyme activity remained constant at a high level until the early pluteus stage. During gastrulation, an increase in the fraction of DNA polymerase-α was associated with the rough endoplasmic reticulum. During the period between the gastrula and pluteus stages, the cytoplasmic DNA polymerase-α activity decreased gradually with a concomitant increase of activity in the nucleus fraction. The timing of this increase in the nucleus coincided with the increase of DNA content per embryo. These results suggest that DNA polymerase-α accumulates on the rough endoplasmic reticulum during gastrulation and then translocates to the nucleus for DNA synthesis as seen before the blastula stage. DNA polymerase-α obtained from gastrula nuclei did not associate with the endoplasmic reticulum from gastrulae. DNA polymerase-α obtained from the gastrula endoplasmic reticulum membranes became bound to the salt-washed membranes from gastrulae but not to those from unfertilized eggs. Likewise, DNA polymerase-α from the rough endoplasmic reticulum of unfertilized eggs became attached to salt-washed membranes from unfertilized eggs, but not to those from gastrulae. This suggests that DNA polymerase-α is synthesized anew, and a transition of both DNA polymerase-α and endoplasmic reticulum occurs at the gastrula stage.     

Ontogeny of T cells: development of pre-T cells from fetal liver and yolk sac in the thymus microenvironment

The patterns of development of T cells from the very early stem cells that settle in the embryonic thymus have been studied. For this purpose, mouse embryonic thymuses (14 days) depleted of thymocytes were reconstituted with hemopoietic stem cells from fetal liver (FL) and yolk sac (YS) and T-cell development was followed in vitro in organ culture. It was found that cells derived from FL and YS of 10- to 14-day-old embryos were capable of reconstituting depleted thymic explants and exhibiting membrane markers in a pattern similar to that of thymocytes developing in intact thymic explants. Furthermore, these cells responded to concanavalin A in proliferative and cytotoxic assays as measured by limiting-dilution analysis. Thus, lymphohemopoietic stem cells emerging in the embryo prior to thymus lymphoid development are capable of differentiation in the thymus microenvironment into T cells, identified by phenotypic markers and functions that are characteristic of cells developing in the intact embryonic thymus.     

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.     

Vascular remodeling of the mouse yolk sac requires hemodynamic force

The embryonic heart and vessels are dynamic and form and remodel while functional. Much has been learned about the genetic mechanisms underlying the development of the cardiovascular system, but we are just beginning to understand how changes in heart and vessel structure are influenced by hemodynamic forces such as shear stress. Recent work has shown that vessel remodeling in the mouse yolk sac is secondarily effected when cardiac function is reduced or absent. These findings indicate that proper circulation is required for vessel remodeling, but have not defined whether the role of circulation is to provide mechanical cues, to deliver oxygen or to circulate signaling molecules. Here, we used time-lapse confocal microscopy to determine the role of fluid-derived forces in vessel remodeling in the developing murine yolk sac. Novel methods were used to characterize flows in normal embryos and in embryos with impaired contractility (Mlc2a(-/-)). We found abnormal plasma and erythroblast circulation in these embryos, which led us to hypothesize that the entry of erythroblasts into circulation is a key event in triggering vessel remodeling. We tested this by sequestering erythroblasts in the blood islands, thereby lowering the hematocrit and reducing shear stress, and found that vessel remodeling and the expression of eNOS (Nos3) depends on erythroblast flow. Further, we rescued remodeling defects and eNOS expression in low-hematocrit embryos by restoring the viscosity of the blood. These data show that hemodynamic force is necessary and sufficient to induce vessel remodeling in the mammalian yolk sac.     

In vitro differentiation of mouse embryonic yolk sac cells

The embryonic yolk sac is the first site in the mammalian embryo in which cells are found that can carry out cell-mediated immune functions, yet the relation of cells of this primitive hematopoietic organ to the development of the mature immune system has not been established. We have initiated a series of experiments to determine the potential of cells of the mouse yolk sac to differentiate in vitro, in order to get an insight into the development of immunocompetence in this primary population of hematopoietic stem cells. The present paper describes the conditions promoting stem-cell differentiation and provides an initial characterization of cell surface phenotypes of the cell lineages established in vitro. Yolk sac cells obtained from 10- to 13-day mouse embryos were maintained in culture for more than 18 months, giving rise to a variety of cell types belonging to the hematopoietic lineages and culminating in the establishment of long-term cell lines. Supernatants of secondary mixed leukocyte cultures were found to be an effective source of growth factors promoting the initial differentiation as well as the maintenance of these cells. Flow-cytometric analysis showed that, in contrast to freshly obtained yolk sac cells, which had no detectable Thy 1 antigen, cells expressing significant levels of Thy 1 were obtained after 1 week or more of culture. Ly1 and Lyt 2 antigens were detected only rarely and the L3T4 (GK 1.5) antigen was never expressed.(ABSTRACT TRUNCATED AT 250 WORDS)     

A comparison of two cloned mouse beta-globin genes and their surrounding and intervening sequences

The BALC/c mouse has two nonallelic beta-globin genes that appear to reside on two different Eco R1 fragments of genomic DNA. We have already cloned one of these fragments and shown that the gene encoded within it is interrupted by at least one large intervening sequence of DNA. We have now cloned and characterized the second beta-globin gene-containing fragment. The coding sequence of its gene is also interrupted by an intervening sequence of DNA that occurs in about the same position, relative to the coding sequence, as does the first. Because some shared features of the structure of these two genes might be responsible for their coordinate expression and the elimination of their intervening sequences, we have compared their surrounding, coding and intervening sequences by restriction endonuclease analysis and by visualization of the heteroduplex structures formed between them. Of the 7000 bp of sequence compared in this way, we find only a few hundred base pairs of homology in addition to the coding sequence. These shared sequences flank the coding sequence and appear to include only those portions of the intervening sequence immediately adjacent to the interrupted structural gene.     

Variant chromosomal arrangement of adult beta-globin genes in rat

The genomic organization of three haplotypes of beta-globin genes was determined to resolve the question of the number of those genes in rat. Haplotype a, found in inbred strain DA, has three genes or pseudogenes, while haplotypes b, found in AO, Y5 and Wistar strains, and c, found in Wistar strain, have five genes or pseudogenes each. In haplotypes b and c, the first gene is of beta major type and the remaining four are of beta minor type. Partial sequencing of six out of 13 genes shows that duplications of beta minor genes are causing polymorphism in a number of genes. Also, in haplotype b two beta minor genes have a 6.5-kb intron 2, while in haplotype c only one beta minor gene contains such a large intron 2. The three structurally different haplotypes described are not interconvertible by single recombination events. The results indicate that the rat has the highest number of adult beta-globin genes found in mammals so far.     

Preferential expression of the maternally derived X chromosome in the mouse yolk sac

We have studied the expression of the maternally derived X chromosome (X^m) and the paternally derived X chromosome (X^p) in female mouse conceptuses on the fourteenth day of gestation. We used an X-linked electrophoretic variant for phosphoglycerate kinase (PGK-1) to estimate the relative proportions of the expression of X^m and X^p in the fetus and in the yolk sac. Our results support the cytological observations of Takagi and Sasaki (1976) and suggest that X^m is preferentially expressed in the mouse yolk sac. Further analysis strongly suggests that the paternally derived Pgk-1 allele (and therefore probably the whole of X^p) is not expressed in the mouse yolk sac endoderm. We have demonstrated that this effect is not caused by a selection pressure exerted by the phenotype of the maternal reproductive tract against cells which express X^p.We therefore, conclude that the parental origin of X^m and X^p marks them as different from one another. Possible causes for the failure of the expression of X^p in the yolk sac endoderm and the tissue specificity of the effect are discussed.