Differential localization of villin and fimbrin during development of the mouse visceral endoderm and intestinal epithelium.

The apical surface of transporting epithelia is specially modified to absorb nutrients efficiently by amplifying its surface area as microvilli. Each microvillus is supported by an underlying core of bundled actin filaments. Villin and fimbrin are two actin-binding proteins that bundle actin filaments in the intestine and kidney brush border epithelium. To better understand their function in the assembly of the cytoskeleton during epithelial differentiation, we examined the pattern of villin and fimbrin expression in the developing mouse using immunofluorescence and immunoelectron microscopy. Villin is first detected at day 5 in the primitive endoderm of the postimplantation embryo and is later restricted to the visceral endoderm. By day 8.5, villin becomes redistributed to the apical surface in the visceral endoderm, appearing in the gut at day 10 and concentrating in the apical cytoplasm of the differentiating intestinal epithelium 2-3 days later. In contrast, fimbrin is found in the oocyte and in all tissues of the early embryo. In both the visceral endoderm and gut epithelium, fimbrin concentrates at the apical surface 2-3 days after villin; this redistribution occurs when the visceral endoderm microvilli first contain organized microfilament bundles and when microvilli first begin to appear in the gut. These results suggest a common mechanism of assembly of the absorptive surface of two different tissues in the embryo and identify villin as a useful marker for the visceral endoderm.

Lectin binding of F9 embryonal carcinoma cells: evidence for population heterogeneity and developmentally regulated high-Mr cell surface proteins.

Undifferentiated F9 embryonal carcinoma (EC) cells bound fluorochrome-coupled Helix pomatia agglutinins (HPA) and peanut agglutinins (PNA) homogeneously, but were distinctly heterogeneous in their binding of Dolichos biflorus agglutinin (DBA) conjugates. Upon chemically induced differentiation the proportion of cells binding the DBA conjugates increased, but a distinct heterogeneity in the intensity of binding remained among the parietal endoderm (PE)-like F9 derivatives. These cells were heterogeneous in their binding of HPA conjugates as well, and many of them failed to bind PNA conjugates, apparently due to sialylation of the PNA-binding sites. Electrophoretic analysis of lectin-binding glycoproteins in the detergent-soluble fraction of the cells revealed the appearance of a doublet of polypeptides of Mr 300,000-400,000 upon differentiation induced by retinoic acid (RA). In addition, an Mr 220,000 polypeptide appeared upon differentiation induced by RA and dibutyryl cyclic AMP (dbcAMP). These polypeptides were obtained from both metabolically labelled and surface-labelled cells. A major secreted glycoprotein, which comigrated with laminin, bound to DBA. This suggests that laminin secreted by the differentiated F9 derivatives contains O-glycosidic saccharides. The results show that even though differentiation of F9 cells leads to changes in their binding of fluorochrome-coupled lectins, these lectin conjugates reveal distinct population heterogeneity among undifferentiated and differentiated F9 cells and are hence likely to be of limited value in the characterization of individual cells. At the whole cell population level, on the other hand, affinity binding to lectins reveals the appearance of high-Mr cell surface proteins in differentiating F9 cells.

Widespread distribution of type II collagen during embryonic chick development.

Type II collagen is a major component of hyaline cartilage, and has been suggested to be causally involved in promoting chondrogenesis during embryonic development. In the present study we have performed an immunohistochemical analysis of the distribution of type II collagen during several early stages of embryonic chick development. Unexpectedly, we have found that type II collagen is widely distributed in a temporally and spatially regulated fashion in basement membranes throughout the trunk of the embryo at stages 14 through 19, including regions with no apparent relationship to chondrogenesis. Immunohistochemical staining with two different monoclonal antibodies against type II collagen, as well as with an affinity-purified polyclonal antibody, is detectable in the basement membranes of the neural tube, notochord, auditory vesicle, dorsal/lateral surface ectoderm, lateral/ventral gut endoderm, mesonephric duct, and basal surface of the splanchnic mesoderm subjacent to the dorsal aorta, and at the interface between the epimyocardium and endocardium of the developing heart. In contrast, immunoreactive type IX collagen is detectable only in the perinotochordal sheath in the trunk of the embryo at these stages of development. Thus type II collagen is much more widely distributed during early development than previously thought, and may be fulfilling some as yet undefined function, unrelated to chondrogenesis, during early embryogenesis.

Reactivity of visceral yolk sac endoderm with lectins: changes related to age and species.

The expression of receptors for lectins reacting with extraembryonal endoderm was compared between mouse and rat at different stages of gestation. Fluorescein-conjugated Helix pomatia agglutinin (HPA), Dolichos biflorus agglutinin (DBA), Sophora japonica agglutinin (SJA), peanut agglutinin (PNA), Bandeiraea simplicifolia agglutinin 1 (BSA-1) and gold-conjugated DBA and PNA were used. It was shown that the rat visceral endoderm does not express the receptors for HPA, while the mouse tissue does. Other lectins react with the visceral endoderm of both species but the reactivity disappears at different days of gestation. The control of expression of receptors for the lectins suggests that they may be involved in the recognition system important during differentiation.

Teratogenic antibody internalization by rat visceral yolk-sac endoderm in vitro: an ultrastructural colloidal gold tracer study.

Previous work from our laboratory has demonstrated that specific rabbit immunoglobulins G (IgG) against a glycoprotein antigen of rat kidney proximal tubule or a cross-reacting visceral yolk-sac endodermal cell antigen will induce abnormal embryonic development when they are injected into pregnant rats during the period of organogenesis. It has been proposed that these antibodies may induce embryopathy by interfering with functions of the visceral yolk-sac placenta, an important organ providing nutrients to the embryo at this stage of development. In order to gain some insight into the underlying pathogenic mechanism(s) in which specific teratogenic IgG may interfere with visceral yolk-sac functions, we examined the uptake of these teratogenic IgG by the visceral yolk-sac endodermal cells at the electron microscopic level. The results demonstrated that teratogenic rabbit IgG specifically localized on the fuzzy coat of the external apical cell membrane of the visceral yolk-sac endoderm at the intermicrovillous region. Within 5 min, the IgG were rapidly internalized via coated pits and micropinocytic vesicles. Within 30 min, an increasing proportion of gold particles appeared within uncoated vesicles or vacuoles of various sizes; most of the gold particles were in close proximity to the inner membranous lining of the vesicles. Similar findings were observed after 1- or 2-hr incubation. After 24- to 48-hr culture, however, the gold particles appeared to have dissociated from the inner surface of the vesicle membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

The outgrowth of parietal endoderm from mouse teratocarcinoma stem-cell embryoid bodies.

Teratocarcinoma stem cells can be used to study certain events occurring during early mouse embryogenesis. We report that the outgrowth of parietal endoderm from teratocarcinoma stem-cell embryoid bodies in vitro is analogous to the same process in vivo in terms of the spatial distribution of endoderm types: only parietal endoderm migrates away from the aggregate, whereas visceral endoderm remains associated with the embryoid body. The outgrowths generated on a substrate of type-I collagen from PSA-1 and retinoic-acid-treated F 9 embryoid bodies were found to be comparable, even though these aggregates express different endoderm types. We demonstrated that retinoic-acid-treated F 9 embryoid bodies that contain essentially only visceral endoderm in suspension culture can nonetheless generate parietal-endoderm outgrowth when plated on type-I collagen, suggesting that substrate interaction plays an important role in inducing parietal-endoderm differentiation. These data indicate the usefulness and relevance of studying endoderm differentiation and outgrowth in vitro employing the teratocarcinoma model system.

[Immunological localization of alpha-fetoprotein in the proximal endoderm and its significance in extra-embryonic differentiation of teratocarcinomas in the mouse]. / Le marquage immunologique de l'alpha-foetoprotéine dans l'endoderme proximal et sa signification dans les différenciations extra-embryonnaires des tératocarcinomes de la souris.

The ABC technique using a specific anti-mouse alpha-foetoprotein (AFP) serum permits identification of groups of extraembryonic proximal endoderm cells in certain teratocarcinomas. These AFP+ cells are always associated with trophoblastic and parietal endodermal patterns and structures. These observations strongly support the hypothesis of a common precursor to the extraembryonic tissues which appears during the first phases of egg development.

Expression of c-fos in parietal endoderm, amnion and differentiating F9 teratocarcinoma cells.

The expression of the cellular proto-oncogene, c-fos, in extra-embryonic tissues of the mouse was investigated using a v-fos DNA probe and an affinity-purified antiserum raised against a C-terminal synthetic peptide. At 13.5 days of development, parietal endoderm--a tissue not previously studied using these methods--was found to express c-fos RNA at a higher level than the amnion or placenta. The previously reported dramatic increase in c-fos RNA levels in extra-embryonic membranes during gestation was found to be confined to the amnion. The antipeptide serum specifically recovered proteins with Mr values of 46,000 and 39,000 from extracts of parietal endoderm and amnion cells labelled for 15 min with 35S-methionine. On sodium-dodecyl-sulphate/polyacrylamide gel electrophoresis these proteins co-migrated with proteins immunoprecipitated using serum from rats inoculated with FBJ-MuSV-transformed cells (tumour-bearing rat serum). Pulse-chasing and 32P-labelling experiments showed that the protein with an Mr of 46,000 was rapidly converted into higher-molecular-weight phosphorylated derivatives. F9 teratocarcinoma stem cells differentiated into parietal-endoderm-like cells in response to treatment with retinoic acid and dibutyryl cyclic AMP. However, this differentiation was not accompanied by any large transient increase in c-fos RNA expression.

Apolipoprotein expression by murine visceral yolk sac endoderm.

Apolipoprotein expression was examined in the postimplantation mouse embryo. Antibodies directed against murine Apolipoprotein AI and human low-density lipoprotein (LDL) particles specifically immunoprecipitated metabolically labelled radioactive apolipoproteins from the culture supernatant of 10.5 days post coitum (days p.c.) yolk sac visceral endoderm cultured in vitro. No evidence for apolipoprotein expression by other embryonic or extraembryonic tissues at this stage was obtained. Immunohistochemical staining at sectioned 10.5 days p.c. embryos with anti-Apolipoprotein AI antibodies revealed specific localization of immunoreactive material in the yolk sac visceral endoderm. We conclude that the yolk sac visceral endoderm is a source of lipoproteins during postimplantation embryonic development.

Cell surface-associated proteins which bind native type IV collagen or gelatin.

Gelatin coupled to Sepharose has been used to isolate [35S]methionine-labeled polypeptides of Mr = 47,000, 56,000, 62,000, and 65,000 from the 12,000 X g supernatant of detergent extracts of mouse embryo parietal endoderm cells. The polypeptides can also be recovered from various established cell lines which synthesize type IV procollagen, and in these cells the Mr = 47,000 polypeptide is the major gelatin-binding component. Several lines of evidence, including the results of continuous labeling and pulse-chase experiments, show that the polypeptides are not derived by proteolytic cleavage of larger precursors and are distinct from fragments of fibronectin. The Mr = 47,000, 62,000, and 65,000 polypeptides all contain N-linked oligosaccharide side chains, as judged by their labeling with [3H]mannose and their sensitivity to tunicamycin. The Mr = 62,000 and 65,000 polypeptides can be resolved by two-dimensional gel electrophoresis into species with different isoelectric points, and the Mr = 47,000 has a pI of 7.5-8.0. None of the gelatin-binding polypeptides appear to accumulate in the culture medium, and the Mr = 47,000, 62,000, and 65,000 species are labeled in a lactoperoxidase-catalyzed iodination of intact cells, suggesting that they are associated with the cell surface. Only the Mr = 47,000 glycoprotein binds to native type IV collagen. Possible functions for these surface components in vivo are discussed.

The identification of a tissue-restricted plasma membrane marker in Xenopus laevis embryos by using a monoclonal antibody.

A library of monoclonal antibodies raised against partially purified membrane fractions from Xenopus laevis oocytes has been produced. One of these antibodies has been cloned and characterized in detail. It was found to be specific for a membrane-bound antigen of apparent Mr, 55,000. The distribution of the antigen has been studied by indirect immunofluorescence on sections of X. laevis embryos and has been found to be highly specific for some ectodermal and endodermal tissues. It was not present on mesodermal tissues.

Procollagen IV. Association to tetramers.

Procollagen IV was isolated from culture media of the mouse endodermal cell line PF-HR9. Some of the triple helical procollagen IV molecules were associated at their NH2 ends to tetramers which were identified by electron microscopy, velocity sedimentation, and electrophoresis. The formation of these tetramers in cell cultures and from isolated procollagen IV molecules was investigated. After an initial noncovalent association, which is reversible, disulfide bonds form between molecules. Even alkylated molecules form disulfide-linked tetramers when exposed to a mixture of reduced and oxidized glutathione. This reaction requires an adequate concentration of procollagen and is not facilitated by added laminin, Ca2+, or Mg2+ ions. Cystine, as a normal constituent of cell culture media, interferes in tetramer assembly, presumably by forming mixed disulfides. Tetramers formed normally and under the influence of glutathione are similar, but probably not identical, and resemble those isolated from fragmented basement membranes. We conclude that the NH2 ends of procollagen IV molecules can associate into tetramers without the help of other molecules and that disulfide bridges subsequently stabilize the association in various ways.

Flow sorting in the study of cell-cell interaction.

Undifferentiated mouse teratocarcinoma cells were cocultivated with differentiated mouse endoderm cells in order to study the possible induction of teratocarcinoma cell differentiation. A difference in DNA content between the two cell types was experimentally introduced to enable the reisolation of the teratocarcinoma cells after cocultivation. Pseudotetraploid (2s) endoderm cell lines were produced from pseudodiploid (1s) cells by treatment of these cells with cytochalasin B and flow sorting of tetraploid cells, using Hoechst 33342 as a viable DNA stain, with subsequent cloning of sorted single cells. In model experiments, where mixtures of 1s teratocarcinoma and 2s endoderm cells were stained with Hoechst 33342, the teratocarcinoma cells could be reisolated with a purity of about 97%. After a cocultivation period of 24 days viable teratocarcinoma cells could be isolated from the cocultivation mixture with a purity of 95%. Two dimensional analysis of the protein pattern of these cells indicated that cocultivation did not induce a differentiated (endoderm) pattern. Therefore according to this analysis the teratocarcinoma cells were not induced to differentiate during a 24 day cocultivation period. The method described offers excellent possibilities for studying cell-cell interaction in vitro.

Differential expression of lectin receptors in germ layers of the mouse egg cylinder and teratocarcinomas.

Receptors for three lectins with restricted specificities, namely fucose-binding protein of Lotus tetragonolobus (FBP), peanut agglutinin (PNA) and Dolichos biflorus agglutinin (DBA), were distinctively located in 6- and 7-day mouse embryos and in embryoid bodies of teratocarcinoma OTT6050 grown in vivo. Thus, FBP reacted mainly with the inner cells (embryonic ectoderm and teratocarcinoma stem cells), DBA reacted with the outer cells (endoderm) and PNA reacted with all the germ layers including mesoderm. Upon in vitro culture of the embryoid bodies, the exposed stem cells express DBA receptors. Since the receptors for the three lectins in teratocarcinomas are known to be carried by the large carbohydrate chains characteristic of early embryonic cells, the present result suggests that terminal structure of the large carbohydrates is altered according to the direction of the differentiation or to the position of the cells in embryos and in teratocarcinomas.

Tissue specificity of alpha-fetoprotein messenger RNA expression during mouse embryogenesis.

Alpha-fetoprotein (AFP) is a synthetic product of only the visceral yolk sac endoderm and fetal liver during mouse embryogenesis. To examine the involvement of transcriptional and post-transcriptional mechanisms in the regulation of tissue-specific expression of the AFP gene, the distribution of AFP mRNA in tissues at different stages of development was determined. Total RNA was isolated from mid-gestation tissues, and a microextraction procedure was developed for earlier stage embryos where the amount of tissue was limited. AFP mRNA was measured by a dot-hybridization assay using a 32P-labelled AFP cDNA clone as probe. Results show that only visceral yolk sac endoderm and fetal liver of midgestation embryos contained AFP mRNA. At earlier stages AFP mRNA was confined to regions within the visceral endoderm which have previously been shown to be AFP positive. These results indicate that expression of the AFP gene during post-implantation development is probably controlled at the level of AFP gene transcription. In addition, we show by in situ hybridization to tissue sections that all endoderm cells of the visceral yolk sac contain AFP mRNA, indicating that the visceral endoderm layer is a homogeneous population of cells with respect to transcription of the AFP gene.

Localization of receptors for Dolichos biflorus agglutinin in early post implantation embryos in mice.

The distribution of receptors for Dolichos biflorus agglutinin (DBA) was studied by histochemical staining of paraffin sections with HRP- or FITC-DBA in mouse embryos at stages ranging from 4.5 to 12.5 days post coitum. Preimplantation blastocysts did not express DBA receptors. The receptors first appeared in primitive endoderm cells in 5-day embryos. In 5.5- to 7.5-day embryos, both the parietal and extraembryonic visceral endoderm cells expressed the receptors. Reichert's membrane was negative for DBA receptors. The columnar cells of the embryonic visceral endoderm (EVE) strongly expressed the receptors, while the flat cells at the antimesometrial pole of the EVE were negative or patchily positive. In 8- to 9-day embryos, the receptors were expressed in the epithelium of the fore- and hindgut. In 9.5- to 12.5-day embryos, the epithelial cells of various regions of the gut expressed the receptors, although the endodermal cells of the liver and the pancreas, both derived from the foregut, did not express them. In 8- to 12.5-day embryos both the visceral and parietal yolk sac endoderm were positive in DBA receptors. The receptors were localized exclusively on the free surface facing the yolk cavity or on the luminal surface and subjacent cytoplasm. All endodermal cells which were positive are known to have absorptive activity. All other tissues in these stages were negative in DBA receptors.