New differentiation markers of mouse liver epithelial cell lines

We describe two new markers of mouse liver epithelial cells detected by monoclonal antibodies. Immunomorphological localization of antigens was performed using light and electron microscopy. Antigen G7 is a marker of cholangiocytes and oval cells. Antigen A6 is present in cholangiocytes and oval cells; moreover, it is expressed in normal liver in single hepatocytes adjacent to the portal vein, in preneoplastic liver, in newly formed hepatocytes, and in certain hepatocarcinomas. Thus, antigen A6 is a marker of cholangiocytes, oval cells and of certain stages of hepatocyte differentiation. We also detected phenotypic heterogeneity of Gehring cells in terms of antigen A6 content. We have formulated problem of the relationship between A6-negative Gehring cells and liver stem cells. Both marker antigens are species-specific but are not specific for the liver. Antigen A6 is simultaneously a differentiation marker of cells belonging to the erythroid series. It is expressed in erythroblasts of fetal liver and is absent in erythroblasts of the yolk sac and erythrocytes. The relationship between antigen A6 and blood group antigens is discussed.     

Common antigens of mouse oval and biliary epithelial cells. Expression on newly formed hepatocytes

Two antigens - A6 and G7 - shared by mouse biliary epithelial and oval cells were revealed by monoclonal antibodies raised in rat immunized with oval-cell-enriched liver fraction. Oval cells were induced in CBA or F1 (CBA x C57BL6) mice by a combination of a single injection of the alkylating drug Dipin with partial hepatectomy. In normal liver A6 antigen was localized, using light and electron microscopy, in biliary epithelial cells of all ducts including Hering canals. Some bile ductal and Hering cells were A6-negative. Occasionally, A6 antigen was present in single hepatocytes forming the periportal ends of hepatic cords. In preneoplastic and tumorous liver A6 antigen was present in bile ductal and oval cells and in a fraction of newly formed hepatocytes and tumor cells. G7 antigen was revealed in normal, precancerous and tumorous liver in biliary epithelial and oval cells but not in hepatocytes. A6 and G7 antigens were not liver-specific: they were expressed in various normal organs and tissues, especially in epithelia. In studies of mouse liver lineages A6 antigen can be used as a common marker of biliary epithelial and oval cells and hepatocytes at certain stages of differentiation. G7 antigen is a marker of oval and biliary epithelial cells. There was a striking similarity in A6 antigen localization to that of human blood group antigens in normal liver and liver tumors. A6 antigen may thus provide a useful tool for the study of neoexpression of human blood group antigens in liver tumors.     

The action of the cells of hemopoietic organs in chick embryos on mouse CFUdc and CLFUdc]

The humoral influence of cells of hemopoietic organs of chicken embryos of different terms on the development of the colony and cluster formation of mononuclears of the bone marrow of mice was studied in joint cultivation in two-compartment cylindrical diffuse microchambers. The process of formation of colonies and clusters is inhibited by cells of the yolk sac on the 2nd-4th day of the development, by cells of the liver on the 8th-12th day, of the spleen on the 13th-18th day and of the bone marrow--on the 15th day. The yolk sac cells were found to have most considerable inhibiting influence on proliferation and differentiation of cells on the 2nd day of the development of chicken embryo. The yolk sac cells on the 6th day stimulate the formation of colonies and clusters. The yolk sac, beginning from the 4th day of the development, and the liver release humoral factors promoting the formation of erythroid colonies. The erythroid colonies are formed but when cultivated on the vascular membrane of the chicken embryo; the erythroid colonies are not formed when cultivated in the abdominal cavity of mice. Local erythropoietinoid factors are not synthetized by the spleen and bone marrow cells. A supposition is put forward that a combination of the local inhibiting and erythropoietic effects promotes the erythroid differentiation of cells.     

On the kinetics of erythroid cell differentiation in fetal mice. II. DNA and hemoglobin measurements of individual erythroblasts during gestation

Microspectrophotometric absorption measurements were used to determine the hemoglobin content of erythroid cells derived from the yolk sac during gestation of fetal C3H mice, from day 9 to day 15. Using the DNA content as a marker for the mitotic state between 2C and 4C phase, five successive cell generations and their mean hemoglobin contents were distinguished: 12 pg (pg, picogram = 10^?12 gm). 22.2 pg, 37 pg, 50 pg and 56 pg. In the final state, nucleated erythrocytes contained 98 ± 22 pg hemoglobin. Erythroid cells derived from the liver were measured on day 15 of fetal gestation. The hemoglobin content of proerythroblasts was below 0.3 pg. The two cell generations in the basophilic state had 0.6 pg and 1.7 pg respectively. Polychromatic erythroblasts yielded a hemoglobin content of 5.1 pg in the first cell generation and 7.5 pg in the second one. Orthochromatic erythroblasts contained 8 pg, reticulocytes 12 pg and mature erythrocytes 28 ± 7 pg hemoglobin. Calculations based on these data suggest that the rate of total hemoglobin synthesis is similar in both yolk sac and liver erythropoiesis. The difference between the final hemoglobin content in nucleated erythrocytes of yolk sac origin and that in hepatic erythrocytes can be explained by the different cell generation times.     

The role of the yolk sac in craniofacial development of cultured rat embryos

To study the role of the yolk sac and amnion in craniofacial development, the effects of opening the yolk sac and amnion on facial formation of rat embryos were examined in vitro. Rat embryos were cultured for 72 hr from day 11.5 of gestation using an improved rotation apparatus. In experiments, the yolk sac and amnion were opened at the time of explantation (day 11.5) in one group (D11 open) and were opened 24 hr after the beginning of the culture (day 12.5) in another group (D12 open). Cleft lip developed in 100% of cultured embryos when the yolk sac and amnion were opened at day 11.5 (D11 open). In the D12 open group, however, cleft lip occurrence decreased to 3.0%. Protein content, wet weight, and somite number of cultured embryos were not significantly different in the two groups. The results of this study demonstrate that it is beneficial to open the yolk sac and amnion after 24 hr in culture for normal facial formation of rat embryo cultured from day 11.5 of gestation.     

Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver

The yolk sac and the para-aortic splanchnopleura/aorta-genital ridges-mesonephros (P-Sp/AGM) region are the main sites of haematopoietic activity in the mouse embryo at the pre-liver stage of development. By day 11.5 of gestation, the AGM region is capable of autonomous initiation and expansion of definitive haematopoietic stem cells (HSCs). By day 12.5, HSC activity in the AGM region is reduced whilst a second wave of HSCs begins to emerge in the yolk sac. We show here that HSCs emerging in both locations are marked by co-expression of the endothelial-specific marker VE-cadherin and the pan-leukocyte antigen CD45. Phenotypic characterisation using CD31, TIE2, FLK1, Ac-LDL receptors, and CD34 markers demonstrated significant similarities between this VE-cadherin+CD45+ ;double-positive' population and endothelial cells suggesting a common origin for these cells. The double-positive fraction also expressed the stem cell markers Kit, Sca1 and AA4.1. Long-term transplantation experiments demonstrated that the double-positive population, which constituted less than 0.05% of the day 11.5 AGM region and the day 12.5 yolk sac, is highly enriched for HSCs. In vitro assays showed that this population is also enriched for myeloid progenitors. During foetal liver colonization, circulating HSCs remained within the VE-cadherin+ cell fraction, although their phenotypic similarity with endothelial cells became less prominent. Upon liver colonisation the majority of HSCs downregulated VE-cadherin, expression of which was completely lost in the adult bone marrow. Partial loss of VE-cadherin expression in HSCs can be observed extra hepatically in the advanced AGM region by E12.5. Similarly, the CD34+KIT+ population in the placenta, recently identified as a reservoir of HSCs, partly lose VE-cadherin expression by E12.5. By culturing isolated E11.5 AGM region and E12.5 yolk sac we show that the developmental switch from a ;primary' VE-cadherin+CD45+ to a more ;advanced' VE-cadherin-CD45+ phenotype does not require contact of HSCs with the liver and is probably a function of developmental time.     

Embryonic macrophages of early rat yolk sac: Immunohistochemistry and ultrastructure with reference to endodermal cell layer

Macrophages are multifunctional cells that participate in numerous biological processes; they actively phagocytose foreign particles and cell debris. Embryonic tissue macrophages are present at early stages of mammalian development; their ontogeny and function is still under investigation. Our study used immunohistochemistry and electron microscopy to investigate early rat yolk sac macrophages using mouse antirat macrophage monoclonal antibodies (mAb) Mar 1 and Mar 3 produced by our laboratory. Mar 3 mAb revealed the first emergence of immature macrophages in the rat yolk sac at fetal day nine coinciding with the beginning of yolk sac haemopoiesis that consisted mainly of erythropoiesis, while Mar 1 mAb detected specifically rat yolk sac macrophages at about the 13th to 14th day of gestation. Immunoreactivity against Mar mAbs was mainly located in the yolk sac endodermal cell layer, which may signify endodermal origin of the yolk sac macrophages. Ultrastructurally mature yolk sac macrophages contained numerous endocytic vesicles or vacuoles, well-developed Golgi saccules and many electron dense granules in their cytoplasm and a number of microvillous projections from the cell surface. After establishment of the circulation between yolk sac and embryo, Mar 3 positive cells were also demonstrated inside fetal undifferentiated mesenchymal tissue at fetal day 12. The study demonstrated the first emergence of immature yolk sac macrophages being among the earliest haemopoietic cells formed in mammalian development. Thus, Mar mAbs managed to detect macrophage differentiation antigens through their development early in the rat yolk sac.     

B cell ontogeny in murine embryo studied by a culture system with the monolayer of a stromal cell clone, ST2: B cell progenitor develops first in the embryonal body rather than in the yolk sac.

A stromal cell clone, ST2, which can support both myelopoiesis and B lymphopoiesis of adult bone marrow was used as an in vitro microenvironment for investigating the ontogeny of the B cell progenitor in murine embryos. The B cell progenitor clonable on an ST2 layer first become detectable in the embryonal body rather than in the yolk sac around day 9.5 of gestation. As soon as it develops in the embryo, it enters the blood circulation and becomes detectable both in the developing fetal liver and the yolk sac of the 10 day embryo. On the other hand, mast cell and polymorphonuclear cell progenitors, which are also generated on the ST2 layer, develop first in the yolk sac and migrate to the fetal liver around day 10-11 of gestation. At the late stage of embryonal development, day 15-16 of gestation, the B cell progenitor enters the femur as vascularization of the femur starts. These results suggest that the localization of the committed stem cells for various hemopoietic cell lineages differs in the early embryo, although the localization of the pluripotent stem cells is yet to be determined.     

Characteristics of murine yolk sac erythroid progenitors and their population expansion in liquid culture

Remarkable differences were found between late erythroid progenitors (CFU-e) in cultures of murine yolk sac cells and those of fetal liver cells with respect to frequency, erythropoietin responsiveness and colony size. Cultures of yolk sac on day 11 of gestation showed a CFU-e population of lower frequency, less sensitivity to erythropoietin and smaller colony size than those from cultures of day 14 fetal liver cells. As the proportion of CFU-e to BFU-e was much lower in yolk sac than that in fetal liver, 48-96 h liquid culture experiments were done with these cells to examine the capacity of their precursors to generate a certain amount of CFU-e subpopulations. The cultures of yolk sac cells produced large numbers of CFU-e which formed some large-sized colonies but those of fetal liver cells generated only a small amount of CFU-e.     

Retinol-binding protein synthesis and secretion by the rat visceral yolk sac. Effect of retinol status

Studies were conducted to explore in rats the role of retinol in the regulation of the synthesis and secretion of retinol-binding protein (RBP) by the visceral yolk sac compared to the liver. Previous studies have shown that in retinol deficiency, hepatic RBP secretion is specifically inhibited, whereas hepatic RBP synthesis rate is unchanged. Retinol-depleted, retinoic acid-supplemented female rats were mated, and maternal liver, fetal liver, and visceral yolk sac were obtained at 14 days of gestation (retinol-depleted group). A group of identically treated, retinol-depleted rats were repleted with retinol on the 14th day of gestation, and the same tissues were collected 6 h later (retinol-repleted group). Normal female rats were used as controls. RBP was assayed by radioimmunoassay and RBP mRNA levels by RNase protection assay using a rat RBP cDNA clone. RBP levels in the visceral yolk sac were elevated 10-fold in the retinol-depleted as compared to the control rats and had declined to near normal values in the retinol-repleted animals. The relative levels of RBP mRNA in the visceral yolk sac were very similar in all three groups of rats. Thus, as in the liver, in the visceral yolk sac retinol deficiency inhibits RBP secretion without altering RBP mRNA levels. In the visceral yolk sac, as in the liver, retinol status appears to regulate RBP secretion specifically, without affecting the rate of RBP biosynthesis.     

Characteristics of ceruloplasmin synthesis in embryogenesis and in the early postnatal period of laboratory white rats

The dynamics of ceruloplasmin content was studied by immunochemical methods in the postimplantation rat embryos and postnatal animals. Ten to twenty two day old embryos contained ceruloplasmin (CP) in yolk sac, serum, and amniotic fluid. The highest CP levels were found in yolk sac. CP concentration profiles were almost identical in the serum and amniotic fluid being the highest on the 12th day (0.26 mg%) and the lowest (0.04) on the 16th day of gestation. CP concentration in the serum increased rapidly up to 3.5 mg% from the 17th day of gestation till the term (22nd day) while remaining at a constant and rather low level in the amniotic fluid. Within 16-18 days after birth, CP concentration in the serum remained at the level of 11 +/- 0.3 mg%. Later on it gradually increased and attained plateau (46-48 mg%) by the time of sex maturity. The maternal serum CP does not penetrate, in the embryo, as can be inferred from the experiments with 125I-CP injected into pregnant rats. Differences in the CP degradation rate and modes were found between the embryos and postnatal rats. It is suggested that CP is initially synthesized by the yolk sac endoderm during organogenesis (10-16 days of gestation) and predominantly by the liver during the foetal period (17-22 days).     

A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis.

The c-myb proto-oncogene encodes a sequence-specific DNA-binding protein. To better understand its normal biological function, we have altered the c-myb gene by homologous recombination in mouse embryonic stem cells. Resulting homozygous c-myb mutant mice displayed an interesting phenotype. At day 13 of gestation these mice appeared normal, suggesting that c-myb is not essential for early development. By day 15, however, the mutant mice were severely anemic. Analysis indicated that embryonic erythropoiesis, which occurs in the yolk sac, was not impaired by the c-myb alteration. Adult-type erythropoiesis, which first takes place in the fetal liver, was greatly diminished in c-myb mutants, however. Additional hematopoietic lineages were similarly affected. These results are compatible with a role for c-myb in maintaining the proliferative state of hematopoietic progenitor cells.     

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     

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)     

Cloning of a new mouse foetal beta-globin mRNA sequence.

A novel globin cDNA recombinant (pFG5) has been isolated from a 14-15 day Porton mouse foetal liver cDNA library. It codes for a beta-like globin mRNA expressed in foetal liver-derived erythroblasts and erythrocytes but not in adult reticulocytes nor in yolk sac derived nucleated erythrocytes. It is also found in Friend cells induced to differentiate by DMSO. The nucleotide sequence of pFG5 confirms that it does not code for the beta major or beta minor globin chains nor the embryonic epsilon Y2 globin chain; but it is identical to the published partial sequence of the epsilon Y3 globin gene over the region of overlap (78 nucleotides).     

Temporal expression profiles of organic anion transport proteins in placenta and fetal liver of the rat

Physiological cholestasis linked to immature hepatobiliary transport systems for organic anions occurs in rat and human neonates. In utero, the placenta facilitates vectorial transfer of certain fetal-derived solutes to the maternal circulation for elimination. We compared the ontogenesis of organic anion transporters in the placenta and the fetal liver of the rat to assess their relative abundance throughout gestation and to determine whether the placenta compensates for the late maturation of transporters in the developing liver. The mRNA of members of the organic anion transporting polypeptide (Oatp) superfamily, the multidrug resistance protein (Mrp) family, one organic anion transporter (OAT), and the bile acid carriers Na(+)-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) was quantified by real-time PCR. The most abundant placental transporters were Oatp4a1, whose mRNA increased 10-fold during gestation, and Mrp1. Mrp1 immunolocalized predominantly to epithelial cells of the endoplacental yolk sac, suggesting an excretory role that sequesters fetal-derived solutes in the yolk sac cavity, and faintly to the basal syncytiotrophoblast surface. The mRNA levels of Oatp2b1, Mrp3, and Bsep in the placenta exceeded those in the fetal liver until day 20 of gestation, suggesting that the fetus relies on placental clearance of substrates when expression in the developing liver is low. Mrp3 immunolocalized to the epithelium of the endoplacental yolk sac and less abundantly in the labyrinth zone and endothelium of the maternal arteries. The placental expression of Oatp1a1, Oatp1a4, Oatp1a5, Oatp1b2, Oat, Ntcp, Mrp2, and Mrp6 was low.     

Characteristics of ceruloplasmin synthesis in mammalian embryogenesis. 2. The yolk sac as the site of the primary expression of the ceruloplasmin gene in rats

It was shown that the omphaloid placenta and, first of all, visceral wall of yolk sac is the site of primary synthesis of ceruloplasmin (CP), whereas the activation of CP synthesis in the liver cells is secondary and is revealed from the 12th day of embryo-genesis. The CP synthesis in the yolk sac cells proved by selective CP localization in the cells of the yolk sac visceral wall and, first of all, in the cells of visceral endoderm on sections stained by the method of indirect immunofluorescence and using the reaction of soluble peroxidase-antiperoxidase complex. A specific CP-mRNA has been revealed in the yolk sac cells which is actively translated in the polyribosomes isolated from the yolk sac and in the cell-free translation system from the rabbit reticulocytes. on the 14th day of embryogenesis CP amounts to ca. 4% of all polypeptides secreted by the yolk sac cells. As the embryogenesis proceeds, the relative rate of CP synthesis progressively decreases in the yolk sac and increases in the liver cells. CP synthesized by the yolk sac cells has a molecular mass of ca. 122 kD. Possible causes of differences between the "embryonic" and "adult" rat CPs are discussed. A suggestion has been put forward that the time of activation of CP synthesis coincides with the yolk sac formation (8-9th days of embryogenesis) and the cells of visceral endoderm are the site of primary expression of the CP gene.     

Tissue-specific and developmental expression of human transthyretin gene in transgenic mice

To analyze the regulation of transthyretin gene expression we have produced transgenic mice by microinjecting cloned human transthyretin genes into fertilized eggs of C57BL/6 mice. The 7.6-kilobase (kb) human transthyretin gene containing about 500 base pairs (bp) in the upstream region was used for microinjection. Seven out of nine transgenic mice had detectable amounts of human transthyretin in serum when analyzed by enzyme-linked immunosorbent assay. Transthyretin mRNA was detected in liver and yolk sac but not in other tissues including brain. The amount of mRNA was variable among transgenic mice and was about one-tenth of mouse endogenous transthyretin mRNA. Human and mouse transthyretin mRNAs were detected in liver of fetus and yolk sac at 13 days of gestation and unlike yolk sac the level of mRNA in liver increased gradually during development and reached the maximum at around 17 days of gestation. Human transthyretin was associated with mouse transthyretin to form tetramers as judged from the dilution curve of enzyme-linked immunosorbent assay and the spur formation in Ouchterlony assay.     

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.     

Ligation of the yolk sac circulation and its effect on the yolk sac ultrastructure and development of the rat fetus]

The effect of an interruption of the yolk sac circulation on the rat visceral yolk sac and the development of the fetoplacental unit was examined in the last third of pregnancy. The yolk sac ischaemia was induced by ligating the blood vessels of the yolk sac stalk which connect the vitelline circulation with that of the fetus. A 3-hour ligature caused an extensive swelling of most cell organelles in the epithelial cells and in the capillary endothelia of the yolk sac. Other structural changes were indicative of a cessation of pinocytosis. A 6-hour ligature resulted in a common increase of cell swelling and in a beginning disintegration of the endothelial cells lining the vitelline capillaries. A 15-hour ligature caused severe ultrastructural cell lesions and macroscopical alterations suggestive of a progressive necrolar finding of a nearly complete loss of the amniotic fluid and the death of the fetus, although the maternal blood flow appeared to be still intact in the placenta.