The localization and synthesis of some collagen types in developing mouse embryos.

The location of type IV (basement membrane)collagen in early post-implantation mouse embryos was examined by immunoperoxidase reactions using a specific immunoglobulin raised against mouse lens capsule collagen. Reaction was positive in the earliest embryos studied--on the fifth day of gestation (the day of detection of the copulation plug is the first day). It was found only in the primitive endoderm adjacent to the blastocoelic cavity. Subsequently in development, strong staining reactions were found in the parietal endoderm, Reichert's membrane and an acellular layer which separates the visceral endoderm of the egg cylinder from the ectoderm. In tenth to eighteenth day visceral yolk sacs, the mesodermal portion was stained, which is consistent with the presence of basement membranes around blood vessels. The endodermal portion of the visceral yolk sac did not react, while small amounts were found in the amnion. By incubation of various embryonic tissues with tritiated amino acids, purification of the biosynthesized secreted collagens and their partial characterization, the differential expression of several collagen genes was detected. Identification of collagen types was made by: reaction with specific antibodies to type I and IV collagens; electrophoretic mobility; sensitivity to reduction and to collagenase; analysis of the proportions of 3-hydroxyproline, 4-hydroxyproline and hydroxylysine; and CNBr peptides. In agreement with the data of Minor et al. (1976a) for the rat, mouse parietal endoderm synthesizes large amounts of type IV collagen. In contrast to their findings, however, the 165,000 molecular weight polypeptide is not converted to one of 100,000 after reduction, alkylation and repepsinization (Dehm and Kefalides, 1978). The endoderm of the visceral yolk sac was shown to be synthesizing primarily type I collagen, while the mesoderm layer of this membrane synthesized both type I and IV collagens. Little or no type IV collagen synthesis was detected in the endoderm of the visceral yolk sac. If it is correct that the visceral endoderm of the early embryo makes a major contribution to the formation of the endoderm portion of the visceral yolk sac, then it is clear that a switch in collagen gene expression must occur as it does so.     

Differentiation of the embryonic disc, amnion, and yolk sac in the rhesus monkey

The inner cell mass of the blastocyst has differentiated into epiblast and hypoblast (primitive endoderm) prior to implantation. Since endoderm cells extend beyond the epiblast, it can be considered that both parietal and visceral endoderm are present. At implantation, epiblast cells begin to show marked evidence of polarity. They form a spherical aggregate with their basal ends toward the basal lamina and apical ends toward the interior. The potential for an internal space is formed by this change in polarity of the cells. No cytological evidence of separation of those cells that will form amniotic epithelium from the rest of the epiblast is seen until a cavity begins to form. The amniotic epithelium is originally contiguous with overlying cytotrophoblast, and a diverticulum remains in this position during early development. Epiblast forms a pseudostratified columnar epithelium, but dividing cells are situated toward the amniotic cavity rather than basally. The first evidence of a trilaminar disc occurs when a strand of cells contiguous with epiblast is found extending toward visceral endoderm. These presumptive mesoderm cells are undifferentiated, whereas extraembryonic mesoderm cells are already a distinct population forming extracellular materials. After implantation, visceral endoderm cells proliferate forming an irregular layer one to three cells thick. Visceral endoderm cells have smooth apical surfaces, but very irregular basal surfaces, and no basal lamina. At the margins of the disc, visceral endoderm is continuous with parietal endoderm and reflects back over the apices of the marginal visceral endoderm cells. This sacculation by visceral endoderm cells precedes pinching off of the secondary yolk sac from the remaining primary yolk sac.     

Dynamic connexin43 expression and gap junctional communication during endoderm differentiation of F9 embryonal carcinoma cells

Gap junctional communication permits the direct intercellular exchange of small molecules and ions. In vertebrates, gap junctions are formed by the conjunction of two connexons, each consisting of a hexamer of connexin proteins, and are either established or degraded depending on the nature of the tissue formed. Gap junction function has been implicated in both directing developmental cell fate decisions and in tissue homeostasis/metabolite exchange. In mouse development, formation of the extra embryonal parietal endoderm from visceral endoderm is the first epithelial-mesenchyme transition to occur. This transition can be mimicked in vitro, by F9 embryonal carcinoma (EC) cells treated with retinoic acid, to form (epithelial) primitive or visceral endoderm, and then with parathyroid hormone-related peptide (PTHrP) to induce the transition to (mesenchymal) parietal endoderm. Here, we demonstrate that connexin43 mRNA and protein expression levels, protein phosphorylation and subcellular localization are dynamically regulated during F9 EC cell differentiation. Dye injection showed that this complex regulation of connexin43 is correlated with functional gap junctional communication. Similar patterns of connexin43 expression, localization and communication were found in visceral and parietal endoderm isolated ex vivo from mouse embryos at day 8.5 of gestation. However, in F9 cells this tightly regulated gap junctional communication does not appear to be required for the differentiation process as such.     

High molecular weight extracellular proteins synthesized by endoderm cells derived from mouse teratocarcinoma cells and normal extraembryonic membranes

Rabbit antiserum raised against teratocarcinoma embryoid bodies reacts with two extracellular, collagenase-resistant glycoproteins, PYS A and B, with molecular weights of approximately 350,000 and 220,000 daltons. The 220,000-dalton protein is distinguishable from fibronectin. The two proteins are synthesized and secreted into the medium in large amounts by the teratocarcinoma-derived parietal endoderm line PYS-1, and by normal parietal endoderm cells from the 10.5-day embryo. There was no detectable synthesis of PYS A and B by normal visceral endoderm cells isolated from the 10.5-day embryo, and only trace amounts of PYS A were synthesized by the teratocarcinoma-derived visceral endoderm line PSA5E and by mesodermal cells isolated from the visceral yolk sac. The two proteins therefore seem to be good biochemical markers for distinguishing parietal from visceral endoderm cells. Synthesis and secretion of PYS A and B could not be detected in undifferentiated embryonal carcinoma cells or in endoderm cells derived from them in the presence of retinoic acid.     

The location and synthesis of transferrin in mouse embryos and teratocarcinoma cells

In early postimplantation mouse development, transferrin synthesis appears to be a marker of visceral endoderm cell types. Transferrin was identified using immunoperoxidase staining, in the proximal (visceral) endoderm of the sixth-day egg cylinder, in some tissues at later stages, and in the visceral yolk sac (VYS) at all stages examined. Since the location of a plasma protein does not necessarily indicate its site of synthesis, the incorporation of labeled amino acids into transferrin was studied. Synthesis could be detected in egg cylinders on the seventh day of gestation onwards and in the VYS at all stages. However, although endoderm was the likely tissue source, its ability to synthesize transferrin after its isolation from the embryo was either much reduced or absent. The data are suggestive of a modulating influence by mesoderm and other cell types on transferrin synthesis in visceral endoderm cells. Three types of endoderm-like cells which are produced by teratocarcinoma embryonal carcinoma (EC) cells were analyzed for transferrin synthesis to assess possible parallels with the embryo. Embryoid bodies from PSA1 EC cells contained some outer endoderm cells which stained for transferrin and others which did not. The endoderm line PSA5E but not PYS-2 synthesized transferrin. The third type of endoderm-like cell (END cells) synthesized very little (OC15S1) or no (PC13 clone 5) transferrin. The conclusion that PSA5E, OC15 END, and some differentiated PSA1 cells have visceral endoderm-like character while PYS-2 reflects parietal endoderm phenotype is in agreement with published data.     

Retinoic acid modulation of transmembrane signaling. Analysis in F9 teratocarcinoma cells

F9 embryonal mouse teratocarcinoma cells were differentiated to a primitive endoderm-like phenotype by retinoic acid and to a parietal endoderm-like phenotype by retinoic acid in combination with dibutyryl cyclic AMP. The secretion of tissue plasminogen activator (tPA) is a characteristic of the cells displaying the differentiated phenotypes. The fundamental question of whether tPA secretion is regulated acutely by G-protein-mediated transmembrane signaling was explored. Cells differentiated to primitive and parietal endoderm demonstrated a rapid tPA response to stimulation by beta-adrenergic agonist (isoproterenol). Adenylyl cyclase activity in response to isoproterenol and GTP, but not forskolin, was greater in primitive and parietal endoderm than F9 stem cells. Both primitive and parietal endoderm cells, but not F9 stem cells, displayed beta-adrenergic stimulation of cyclic AMP accumulation. Retinoic acid induced F9 stem cells to the primitive endoderm phenotype and increased beta-adrenergic receptor levels 3-fold. Gi alpha 2 levels declined, G beta-subunits increased, and Gs alpha levels were unchanged following differentiation to primitive endoderm. In parietal endoderm cells beta-adrenergic receptors increased 2-fold over F9 stem cells, Gi alpha 2 levels declined even further than in primitive endoderm, G beta-subunits increased compared to F9 stem cells, and Gs alpha levels again were unchanged. The marked potentiation of short-term stimulation of tPA secretion in the differentiated state may be best explained by the retinoic acid-induced increase in expression of beta-adrenergic receptors coupled with a decline in Gi alpha 2 levels. Short-term regulation by G-protein-linked receptors represents a novel mode for the control of tPA secretion.     

Induction of alpha-fetoprotein synthesis in differentiating F9 teratocarcinoma cells is accompanied by a genome-wide loss of DNA methylation.

F9 teratocarcinoma cells can be grown as monolayers or aggregates, and upon treatment with retinoic acid they will differentiate into parietal or visceral endoderm, respectively. Visceral endoderm specifically synthesizes alpha-fetoprotein and albumin mRNAs, which are not found in parietal endoderm. In contrast, both endoderms produce enhanced levels of the major histocompatibility antigen (H2) mRNA compared with F9 cells. F9 cells contain highly methylated DNA as judged by restriction enzyme digestion. However, upon differentiation into visceral endoderm, there is a genome-wide loss of methylation in induced, silent, and constitutively expressed genes. Experiments in which methylation loss is induced via the methyltransferase inhibitor 5-azacytidine result in no induction of alpha-fetoprotein mRNA and no morphological differentiation, suggesting that methylation loss alone is not sufficient to induce the visceral endoderm phenotype. Likewise, 5-azacytidine treatment of differentiated cells does not result in enhanced expression of alpha-fetoprotein mRNA. However, the patterns of loss of DNA methylation at all sites examined after differentiation or 5-azacytidine treatment were remarkably similar, suggesting that the two occur by a similar mechanism, the inhibition of DNA methyltransferase activity. These results argue that the specificity for methylation loss at a given site is an inherent property of aggregated F9 cell chromatin. This system provides a model for studying a tissue-specific change in DNA methylation upon differentiation.     

Localization and synthesis of the tumor protein oncomodulin in extraembryonic tissues of the fetal rat

The calcium-binding protein oncomodulin, previously found only in tumors, has been detected during rat development. Specific antisera to purified rat hepatoma oncomodulin (MW 11,500) were used to detect oncomodulin by radioimmunoassay (RIA) and by avidin-biotin-peroxidase complex (ABC) immunohistochemistry. Using RIA, oncomodulin was found to increase in placenta from below the limits of detection (2 ng/mg protein) on Day 13 to approximately 25 ng/mg on Day 16 of pregnancy, and to remain high through to the end of gestation. Determinations on separated inner and outer placenta showed the increase to be greater in the outer placenta (basal zone and decidua) than in the inner placenta (labyrinth). The ABC technique on paraffin sections produced positive staining for oncomodulin throughout the placenta, with the most intense staining occurring in the outer placenta (cytotrophoblast and giant cells of the basal zone). Parietal and visceral yolk sac, and amnion also stained positively, while fetal organs did not. Oncomodulin synthesis measured by [35S]methionine incorporation into immunoprecipitates occurred in isolated inner and outer placenta, whole placenta, the separated trophectoderm and endoderm of the parietal yolk sac, and amnion. No oncomodulin synthesis could be measured in visceral yolk sac, fetal liver, or 16-day embryo. This occurrence in developing and transformed tissues demonstrates that oncomodulin is an oncodevelopmental protein.