Characterization of mouse liver alpha-L-fucosidase. Demonstration of unusual basic isoelectric forms of the enzyme that appear to be developmentally regulated.

Mouse tissues contain unusual basic isoelectric forms of alpha-L-fucosidase (with approximate isoelectric points of 8.3 and 9.0) in addition to the usual acidic and neutral forms previously described in tissues of other species. These unusual forms are very prominent in placenta and foetal tissues and comprise approx, 50-80% of total activity up to 11 days of postnatal development. By 15 days of postnatal development, the basic forms are diminished in amount and comprise not more than 25% of total activity. Neuraminidase treatment of adult mouse liver alpha-L-fucosidase led to significantly decreased amounts of acidic forms and increased amounts of the basic forms, suggesting that these forms are chemically related at least in part by sialic acid residues. Comparative kinetic studies on mouse liver, human liver and mouse placental alpha-L-fucosidases indicated that they have the same Km (0.05-0.06 mM) for 4-methylumbelliferyl alpha-L-fucopyranoside but different pH optima and thermostability properties. Mouse liver alpha-L-fucosidase has one pH optimum (5.5) and an acidic shoulder (centred around pH 4.0) compared with two distinct optima (4.3 and 6.8) for the human liver enzyme. Mouse placental alpha-L-fucosidase has a pH-activity curve comparable with that of the mouse liver enzyme except that the acidic shoulder is absent. Mouse liver alpha-L-fucosidase is considerably more thermolabile after preincubation at 50 degrees C than are the human liver and mouse placental enzymes, which gave similar thermodenaturation curves. Immunochemical studies indicated that mouse and human alpha-L-fucosidases are dissimilar antigenically but exhibit some cross-reactivity. The IgG fraction of antibody prepared in goat against human liver alpha-L-fucosidase was ineffective by itself in immunoprecipitating mouse liver alpha-L-fucosidase, but 63% and 72% of the mouse liver and placental enzymes respectively could be immunoprecipitated in the double-antibody experiments under conditions that immunoprecipitated 92% of the human liver enzyme.     

Distinct antigenic characteristics of murine parietal yolk sac laminin

Two monoclonal antibodies (LAM-A and LAM-B) specific for laminin from normal and neoplastic parietal yolk sac (PYS) cells were produced in rats immunized with a mouse yolk sac carcinoma cell line. Both antibodies immunoprecipitated the 400,000- and 200,000-Da chains of laminin and reacted with purified PYS laminin in ELISA. LAM-A reacted with mouse and rat PYS laminin, whereas LAM-B reacted only with mouse PYS laminin. Formaldehyde- and methanol-fixed adult and fetal somatic tissues were immunohistochemically unreactive with either of the two antibodies. In acetone-fixed tissue sections, both antibodies reacted weakly with some medullary tubules of the kidney, the follicular basement membrane of the ovaries, and the seminiferous tubules. The antibodies appear to react with the polypeptide determinants residing on the terminal portion of the long arm of laminin. It is concluded that laminin derived from normal or malignant PYS cells has distinct antigenic sites that are immunochemically not apparent in most other basement membranes.     

Of mice and men: teratomas and teratocarcinomas

Teratomas and teratocarcinomas are tumors containing tissue derivatives of all three germ-layers. They can be induced by transplantation of animal embryos to ectopic microenvironment. Development of malignant teratocarcinomas depends on embryonic stage, species-specificity and immunological competence of the host. In the man, teratomas and teratocarcinomas usually represent a subtype of germ-cell tumors but sacrococcygeal teratomas arise from the remnants of the pluripotent primitive streak. Undifferentiated embryonal carcinoma (EC) cells are responsible for the malignancy of experimental mouse teratocarcinomas. Mouse EC cells injected to the adult give rise to tumors and upon injection to early embryos to differentiated tissues--thus resembling normal mouse embryonic stem cells (mESC). Epigenetic changes rather than mutations are associated with transformation of mESC to EC cells. Human EC and ES cell-lines (hESC) contain chromosomal abnormalities and can form teratocarcinoma after transplantation. ES cells are among those proposed for cell replacement therapy in the man. Suicide gene introduction should be recommended prior to their use in vivo to ablate them in case of malignant transformation.     

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.     

Sequential morphological study of teratomas derived from displaced yolk sac.

The sequential histological and ultrastructural morphology of benign teratomas derived from displaced visceral yolk sac is described. The appearance and the differentiation of all the tissues formed in these teratomas is compared to the differentiation of normal embryonic tissues and to the development and differentiation observed in experimental teratocarcinomas. The presence in the teratomas of adult well-differentiated tissues derived from all three germ layers is discussed in relation to the multipotentiality of cells from the extra-embryonic parts of the embryo.     

Analysis of cell-differentiation lineage in human teratomas using new monoclonal antibodies to cytostructural antigens of embryonal carcinoma cells

Human embryonal carcinoma cells sometimes display the developmental potential of early embryonic stem cells. While available data do not clearly identify a counterpart of these tumor cells in normal development, previous comparisons of human embryonal carcinoma and yolk sac carcinomas indicated that these cell types are closely related, and suggested that embryonal carcinoma cells might resemble the progenitors of extraembryonic endoderm. To analyse further cell-differentiation lineage in these tumors, we produced monoclonal antibodies to cytostructurally associated antigens of human embryonal carcinoma cells. Spleen cells from mice immunized with a detergent-insoluble extract of cultured human embryonal carcinoma cells were fused to NS-1 myeloma cells, and hybridoma supernatants were screened by indirect immunofluorescence on the immunizing cell line, then on a panel of cell lines derived from human embryonal carcinomas, yolk sac carcinomas, and a range of neoplastic and normal tissues. Monoclonal antibody GCTM-1 stained the nuclei of all human cells tested and served as a positive control; this antibody immunoprecipitated proteins of 85 and 66 k Da from human embryonal carcinoma cells. GCTM-2 recognized an epitope on a 200-k Da extracellular protein present on the surface of embryonal carcinoma cells, and stained the surface of visceral yolk sac-type carcinoma and colorectal carcinoma cells as well. Enzymatic analysis of carbohydrate residues on the GCTM-2 antigen revealed that it was a keratan sulphate proteoglycan, and suggested that the epitope recognized by the antibody lies on the core protein. In immunoblots, antibody GCTM-3 bound to a 57-k Da cytoskeletal protein expressed in human embryonal carcinoma. This antibody decorated filamentous arrays in cell lines from human embryonal carcinoma, visceral yolk sac carcinoma, parietal yolk sac carcinoma (endodermal sinus tumour), and adenocarcinoma and large cell carcinoma of the lung. Antibody GCTM-4 recognized a determinant present on a 69-k Da polypeptide, associated with a component of the lysosomal compartment, which was expressed in embryonal carcinoma cells, but no other cell type tested. The results with this antibody panel thus allow distinction between human embryonal carcinoma and yolk sac carcinoma, but provide further evidence of a close relationship between these cell types.     

Isolation and characterization of a multipotent clone of human embryonal carcinoma cells

Histopathological studies suggest that the stem cells of human teratomas may be classified into two major categories: nullipotent stem cells, and multipotent stem cells, capable both of self-renewal and differentiation into a wide range of somatic and extraembryonic cell types. We have isolated a multipotent stem cell clone from the human teratoma cell line GCT 27, and compared its properties to a nullipotent clone derived from the same strain. The multipotent clone GCT 27 X-1 gave rise to colonies of mixed cell morphology in vitro. Analysis of cell surface, cytostructural and extracellular matrix markers in GCT 27 X-1 cells showed that the stem cells of this line were very similar in phenotype to nullipotent cells. The two cell clones were predominantly hypotriploid, and contained several marker chromosomes in common. GCT 27 X-1 was feeder-cell-dependent for continuous growth in vitro; removal of the feeder layer resulted in differentiation of the stem cells into a variety of cell types, some with characteristics of extraembryonic endoderm, others showing neuronal properties. When transplanted into nude mice, GCT 27 X-1 cells gave rise to teratocarcinomas containing embryonal carcinoma stem cells, and many other cell types: yolk sac carcinoma cells; cells producing alphafetoprotein or human chorionic gonadotrophin; glandular, columnar, cuboidal, and squamous epithelium; primitive mesenchyme and cartilage; neuroectodermal cells. Nullipotent GCT 27 C-1 cells could form colonies in the absence of feeder layers, but multipotent GCT 27 X-1 cells could not. While a range of known growth factors and related substances failed to substitute for feeder layers in supporting the growth of GCT 27 X-1 stem cells, supernatants from yolk sac carcinoma cell line GCT 44 could partially replace the feeder cell requirement. Thus, the results revealed a basic difference in growth control between these multipotent and nullipotent human embryonal carcinoma cells, and suggested a possible paracrine regulatory pathway between multipotent stem cells and yolk sac carcinoma cells.     

Neoplastic differentiation: characteristics of cell lines derived from a murine teratocarcinoma

Monolayer cultures of a mouse teratocarcinoma were established in vitro. These cultures contained embryonal carcinoma, the malignant stem cell, and its differentiated progeny: parietal yolk sac, neuroepithelial, and mesenchymal cells. Tissues such as squamous epithelium, cartilage, striated muscle, neuroepithelium, and glands were produced from embryonal carcinoma that was maintained under conditions of long term culture. Frequent subcultivation with pancreatin allowed the establishment of cell lines of embryonal carcinoma which have been maintained for more than 18 months in vitro and continue to produce differentiated cells under specific culture conditions. Chromosomally these lines of embryonal carcinoma have a stem line of 39 chromosomes. Two lines of parietal yolk sac cells have been established which produce basement membrane, are not tumorigenic, and chromosomally are hypotetraploid. This system may yield information concerning neoplastic differentiation and its possible use in therapy for cancer.     

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.     

Differentiation capabilities of human germ cell tumors

It is well known that human germ cell tumors are an excellent model to study not only differentiation capacity of tumor cells but also human normal somatic cell differentiation. A variety of polyclonal and monoclonal antibodies were developed against cell surface antigens of murine embryos and teratocarcinomas. Accumulated data has revealed that these antigens are sequentially expressed on embryonic cells in a well-programmed manner. They have also been shown to be useful markers to investigate somatic cell differentiation in fetal and adult tissue. In humans, however, little is known about the cellular differentiation mechanism in early embryos and whether they could be studied, i.e. whether they occur in human germ cell tumors. In present review, we discussed newly established monoclonal antibodies which were raised from human embryonal carcinoma cells. We have been studying differentiation capacity of human germ cell tumor cells by using these antibodies. Some of these antibodies clearly indicates their usefulness to specify the developmental stage of normal tissue.     

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

We have carried out a comparative analysis of the expression of the albumin and alpha-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 R0t 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.     

Apolipoprotein B-related gene expression and ultrastructural characteristics of lipoprotein secretion in mouse yolk sac during embryonic development.

In mice, the yolk sac appears to play a crucial role in nourishing the developing embryo, especially during embryonic days (E) 7;-10. Lipoprotein synthesis and secretion may be essential for this function: embryos lacking apolipoprotein (apo) B or microsomal triglyceride transfer protein (MTP), both of which participate in the assembly of triglyceride-rich lipoproteins, are apparently defective in their ability to export lipoproteins from yolk sac endoderm cells and die during mid-gestation. We therefore analyzed the embryonic expression of apoB, MTP, and alpha-tocopherol transfer protein (alpha-TTP), which have been associated with the assembly and secretion of apoB-containing lipoproteins in the adult liver, at different developmental time points. MTP expression or activity was found in the yolk sac and fetal liver, and low levels of activity were detected in E18.5 placentas. alpha-TTP mRNA and protein were detectable in the fetal liver, but not in the yolk sac or placenta. Ultrastructural analysis of yolk sac visceral endoderm cells demonstrated nascent VLDL within the luminal spaces of the rough endoplasmic reticulum and Golgi apparatus at E7.5 and E8.5. The particles were reduced in diameter at E13.5 and reduced in number at E18.5;-19.The data support the hypothesis that the yolk sac plays a vital role in providing lipids and lipid-soluble nutrients to embryos during the early phases (E7;-10) of mouse development. secretion in mouse yolk sac during embryonic development.     

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 x C57BL/6)F1 (hereafter called WBB6F1)- +/+ embryos were injected directly into the skin of adult WBB6F1-W/Wv 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 x 10(6)) rescued the general mast-cell depletion of WBB6F1-W/Wv 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.     

The ontogeny of expression of murine metallothionein: comparison with the alpha-fetoprotein gene

The ontogeny of expression of mouse metallothionein was studied by RNA dot and Northern blot hybridization using a cloned cDNA probe. In some instances the synthesis of metallothionein was analyzed by cell-free translation of RNA as well as pulse-labeling of proteins in short-term organ cultures followed by polyacrylamide gel electrophoresis. Interesting parallels between metallothionein and alpha-fetoprotein gene expression during development were noted. Like alpha-fetoprotein mRNA ( Dziadek and Andrews, 1983), metallothionein mRNA was found to be abundant in developing liver as well as in visceral yolk sac endoderm. In addition, metallothionein mRNA was abundant in parietal yolk sac. During liver development metallothionein and alpha-fetoprotein mRNAs were abundant by Day 12 of gestation, increasing to maximal levels on Day 16 and decreasing during late fetal and neonatal life to basal levels in adult. Metallothionein mRNA increased in maternal liver and was also abundant in certain hepatomas. Synthesis of metallothionein and levels of metallothionein mRNA in visceral yolk sac increased from Day 9 of gestation to maximal levels on Days 11-12 and then decreased abruptly after Day 15. RNA from differentiated teratocarcinoma cells with primitive, parietal or visceral endoderm characteristics each contained high levels of metallothionein mRNA, whereas, levels of this mRNA varied widely among embryonal carcinoma stem cell lines. alpha-Fetoprotein mRNA was not detected in embryonal carcinoma cells but was expressed in visceral endoderm-like differentiated cells. These results indicate that parietal and visceral endoderm cells actively express the metallothionein gene and further suggest that expression may be initiated at the earlier stage of primitive endoderm.     

Teratoid Tumors Derived from Mouse Embryos Grown in an Immunologically Privileged Site

Mouse early embryos and embryo fragments were transplanted into an immunologically privileged site, consisting of a glass cylinder previously implanted under the skin of adult mice in order to test their tumor producing potential, in allogeneic adult recipients. The highest yield of tumors was obtained upon transplantation of 6 1/2 day old embryos in toto. i.e., including the embryonic and extraembryonic areas. Histological examination showed teratomas composed of differentiated tissues derived from the three germ layers containing isolated foci of undifferentiated cells and nodules of trophoblast giant cells. Areas exhibiting the histological appearance of yolk sac carcinoma were also observed. Transplantation of the whole 6 1/2 day old egg cylinder, including the ectoplacental cone, and the isolated embryonic area produced a lower incidence of teratomas with a reduced variety of differentiated tissues. No yolk sac carcinoma was found in these grafts. The ectoplacental cone of 6 1/2 day embryos produced no tumors. Grafts of genital ridges from 12 1/2 day embryos gave rise to teratomas with well differentiated tissues of embryonic and extraembryonic origin. Areas ressembling yolk sac carcinoma were also observed. The life span of trophoblastic giant cells within the glass cylinder was significantly longer than in other experimental systems.