Localization and expression of integrin subunits in the embryoid bodies of F9 teratocarcinoma cells

F9 embryonal carcinoma cells can differentiate in vitro into either parietal (PE) or visceral (VE) endoderm, depending upon specific retinoic acid (RA) treatment and growth conditions. In differentiated aggregates of F9 cells (EB), the VE is a polarized monolayer surrounding a core of undifferentiated cells. Within 7 days of treatment the cells organize their cytoskeleton and synthesize large amounts of extracellular matrix proteins to form a basal lamina under the newly formed epithelium. All these changes are likely to involve integrin expression and organization. In this study we have analyzed the spatio-temporal changes in the pattern and level of expression of beta1, beta4, alpha5, alpha6A, and alpha6B integrin subunits. We found that the organization of the VE monolayer in F9 aggregates involves both qualitative and quantitative changes in integrin expression. beta1 is downregulated and accumulates in the forming epithelium. The same occurs for alpha5, although its location on the surface of the aggregate appears to be transient as in fully differentiated EB its distribution is uniform. beta4 and alpha6A are also mainly localized in the VE but they are undetectable in undifferentiated aggregates and their expression is induced by RA treatment. An important exception is represented by alpha6B whose distribution and expression remain almost unchanged throughout treatment.     

The role of extracellular matrix in the migration and differentiation of parietal endoderm from teratocarcinoma embryoid bodies

Embryoid bodies formed from teratocarcinoma stem cells differentiate an outer layer consisting of parietal and visceral endoderm or of visceral endoderm exclusively. We have previously shown that when these embryoid bodies are plated on collagen-coated substrates a parietal endoderm-like cell migrates onto the substrate, whereas all of the visceral endoderm remains associated with the stem cell mass, suggesting a role for substrate contact in parietal endoderm differentiation. We now identify fibronectin as the migration-promoting component in these cultures, and note that laminin and collagen type IV are 10-fold less effective at promoting both attachment and endoderm outgrowth. The RGDS tetrapeptide (arg-gly-asp-ser) from the cell attachment domain of fibronectin can specifically block attachment and outgrowth on both fibronectin- and laminin-coated substrates. In addition, the involvement of the 140-kD fibronectin receptor is demonstrated using an antibody directed against this molecule.     

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.     

Localization of phosphatase activities on mouse teratocarcinoma embryoid bodies

Non-specific alkaline phosphatase and Mg²⁺-dependent adenosine triphosphatase activities were ultracytochemically investigated on embryoid bodies of murine teratocarcinomas, in order to find markers of endodermal cell differentiation of early embryonic cells. The former was localized mainly on the cell surface of inner embryonal carcinoma cells, as already shown by other workers, and weakly on the bound surface of outer endodermal cells of embryoid bodies. The latter, however, was found only on the outer free surface of endodermal cells and never on the surface of embryonal carcinoma cells. It suggests that Mg²⁺-dependent ATP activity might become the marker for early differentiation of embryonal carcinoma cells.     

Moesin signalling induces F9 teratocarcinoma cells to differentiate into primitive extraembryonic endoderm

The mouse F9 teratocarcinoma cell line is a model that can be manipulated to imitate one of the earliest epithelial-mesenchymal transitions in mouse development. When cells are treated with Retinoic Acid they differentiate into primitive endoderm and into parietal endoderm with the addition of dibutyryl cAMP. Parietal endoderm also develops when undifferentiated cells express a constitutively active (CA) form of Galpha13(Q226L). Differentiation is accompanied by a translocation of beta-catenin to the nucleus and considerable changes to the cytoskeleton and cell morphology. ERM proteins facilitate rearrangements to the F-actin cytoskeleton, and at least one, moesin, is essential for cell survival. In this study we found that moesin translocated to the nucleus during RA-induced differentiation, and sequence analysis identified putative nuclear localization signals in the protein. In the absence of RA, transient over-expression of rat moesin or the distantly related zebrafish homologue in F9 cells induced primitive endoderm. Furthermore, no apparent beta-catenin was seen in the nucleus of cells over-expressing zebrafish moesin. Our previous results have shown that depleting F9 cells of moesin using an antisense morpholino strategy caused them to detach from the substrate unless they expressed CA-Galpha13(Q226L). This CA-Galpha13 signalling maintained cell survival, but at the expense of differentiation. We now report that over-expressing zebrafish moesin in mouse moesin-depleted F9 cells not only ensured cell survival, but also induced differentiation to primitive endoderm. Together, the results suggest a new role for moesin, acting in a signalling pathway facilitating the differentiation of extraembryonic endoderm.     

Nucleolus-like bodies in embryonal carcinoma cells of the embryoid bodies isolated from mouse teratocarcinoma

Undifferentiated embryonal carcinoma cells (EC cells) in the embryoid bodies isolated from mouse teratocarcinoma contained nucleolus-like bodies (NLBs) of smaller sizes in their cytoplasm (their sectional area averaged about 0.036 μm²). At the onset of EC cell differentiation, the average sectional area of NLBs significantly increased (about 0.107 μm²). When EC cells had differentiated into mesenchymal cells and endothelial cells of primitive blood vessels, NLBs decreased dramatically both in size and number. The possible role of NLBs in the differentiation process of EC cells is discussed.     

Paracrine promotion of cardiomyogenesis in embryoid bodies by LIF modulated endoderm

In the vertebrate embryo the heart is the first organ to form. Embryonic and extra-embryonic tissues are supposed to contribute to cardiac lineage commitment before and during gastrulation in a paracrine fashion. Evidence has accumulated that factors secreted by the anterior lateral endoderm and extra-embryonic endoderm contribute to cardiomyogenesis. Here we exploit in vitro differentiation of embryonic stem cells in embryoid bodies to study differentiation of the extraembryonic endodermal lineage, gastrulation-like processes, and the influence of endoderm on cardiomyogenesis. We demonstrate that in embryoid bodies primitive endoderm differentiates to visceral and parietal endoderm and that parietal endoderm influences onset of cardiomyogenesis in a concentration-dependent manner. Both increased concentrations of leukemia inhibitory factor and its absence in lif-/- embryoid bodies hampered parietal endoderm formation. Reduced differentiation of parietal endoderm correlated with an attenuation of cardiomyogenesis even in the presence of LIE These and previous results suggest that leukemia inhibitory factor is directly and indirectly, via endoderm formation, involved in the regulation of cardiomyogenesis. Increased proliferation of parietal endoderm in lifr -/- embryoid bodies and addition of conditioned lif -/- cell culture supernatant promoted cardiomyogenesis, demonstrating for the first time that parietal endoderm also contributes to cardiomyogenesis in embryoid bodies in a paracrine and leukemia inhibitory factor and its receptor independent pathway. New factors signaling independently of the leukemia inhibitory-factor receptor pathway may sustain cardiomyocyte cell proliferation and thus be a future target for gene therapy of cardiomyopathies and cell therapy of the myocardium.     

Indian hedgehog signaling in extraembryonic endoderm and ectoderm differentiation in ES embryoid bodies

We previously demonstrated that a member of the Hedgehog gene family, Indian hedgehog (Ihh), is expressed in the visceral endoderm of EC and ES cell embryoid bodies and mouse embryos. Overexpression studies suggested that Ihh was involved in visceral endoderm differentiation. We now provide evidence for a Hh response in the embryoid body core and in the mesothelial layer of the visceral yolk sac. We also demonstrate that treatment of ES embryoid bodies with the Hh antagonists cAMP and forskolin results in downregulation of the Hh response and altered embryoid body differentiation. The outer endoderm layer undergoes a transition to parietal endoderm while formation of an embryonic ectoderm layer surrounding a cavity is inhibited. These treatments also result in a decrease in the expression of markers for the mesoderm derivatives, blood and endothelial cells. We present a model to explain how Ihh and BMP signaling may regulate extraembryonic endoderm and embryonic ectoderm differentiation.     

Induction of the expression of retinol-binding protein and transthyretin in F9 embryonal carcinoma cells differentiated to embryoid bodies

Studies were conducted to determine if the expression of the gene for retinol-binding protein (RBP) and/or transthyretin (TTR) could be induced upon differentiation of F9 teratocarcinoma cells to either visceral endoderm or parietal endoderm. Both TTR mRNA and RBP mRNA were undetectable in the undifferentiated F9 stem cells and in F9 cells differentiated to parietal endoderm. However, TTR mRNA and RBP mRNA were both detected in F9 cell aggregates differentiated to embryoid bodies (which contain visceral endoderm-like cells) by treatment of the aggregates in suspension with retinoic acid. TTR mRNA was observed at 3 days, and RBP mRNA at 5 days, after treatment of the F9 cell aggregates with retinoic acid. Both TTR mRNA and RBP mRNA were found to be specifically localized by in situ hybridization in the outer layer of cells (the visceral endoderm-like cells) of the embryoid bodies. Finally, synthesis and secretion of both RBP and TTR by F9 cell embryoid bodies was demonstrated by specific immunoprecipitation of each newly synthesized protein from the culture medium. These data thus demonstrate the production and presence of RBP mRNA and TTR mRNA, and the synthesis and secretion of RBP and TTR, by F9 cell embryoid bodies (specifically by visceral endoderm-like cells). This finding suggests that these two proteins may be synthesized by rodent embryos extremely early in embryonic development.     

The development of cystic embryoid bodies in vitro from clonal teratocarcinoma stem cells

Previous findings on exogenous RNA-induced heart muscle differentiation in the non-heartforming cultured explants of the chick blastoderm, the postnodal pieces, were reexamined. Some of the changes that characterized the transition in the host tissues were: (i) the formation of highly ordered myofibrils; (ii) the appearance of characteristic cytoplasmic glycogen particles; (iii) a 2.5- and 3.5-fold increase in actin and myosin-like polypetides respectively; (iv) an increase in acetylcholinesterase activity; and (v) the acquisition of spontaneous and rhythmic pulsations. These specific changes appeared only in those explants that received a poly(A)-containing RNA fraction obtained from the 16-day-old chick embryonic heart. Neither synthetic polynucleotides nor a variety of RNA from several sources could replace the RNA from chick embryonic heart as an inducer of heart muscle differentiation.     

Plasminogen activator in early embryogenesis: Enzyme production by trophoblast and parietal endoderm

We have surveyed the early stages in the development and differentiation of cultured mouse embryos for plasminogen activator production. This enzyme is first detectable by the sixth equivalent gestation day. Thereafter, cultured blastocysts produce plasminogen activator with a biphasic time course: in the first phase, enzyme secretion rises to a maximum at about the eighth day and then decreases; a second phase, during which more enzyme accumulates, begins somewhat later and continues to at least the fifteenth day.By fractionating the blastocyst into its constituent cell types, we have identified the trophoblast as the cells responsible for the first phase of enzyme synthesis. The pattern of enzyme production by the trophoblast is closely correlated with the invasive period of these cells in vivo and implies that plasminogen activator is involved in embryo implantation. The second phase of plasminogen activator production is due to parietal endoderm, which initiates enzyme synthesis upon differentiation from the inner cell mass. The properties of the parietal endoderm suggest that plasminogen activator may participate in the migration of these cells and/or in the metabolism of Reichert's membrane which accompanies embryo growth.These results are consistent with the concept, developed from work on other cell types, that plasminogen activator may represent a generalized mechanism for tissue remodeling and cell migration.     

Differential expression of fetomodulin and tissue plasminogen activator to characterize parietal endoderm differentiation of F9 embryonal carcinoma cells.

Fetomodulin is a surface marker protein of differentiated F9 embryonal carcinoma cells. Gene cloning has recently identified it as thrombomodulin which binds thrombin and proteolytically activates protein C. Activity assays and RNA blotting were adopted to analyze F9 cell differentiation with specific reference to another well-characterized marker, tissue plasminogen activator. Retinoic acid induced primitive endoderm differentiation of F9 cells and simultaneously activated tissue plasminogen activator synthesis. This differentiation, however, did not result in fetomodulin expression. When primitive endoderm cells were exposed to 1 mM dibutyryl cyclic AMP, the tissue plasminogen activator level rose further within 6 hr. In contrast, the cofactor activity of fetomodulin stayed below a detectable level for as long as 15 hr and then increased with time. Expression of the two marker proteins appeared to be regulated differently.     

Effect of ascitic liquid on growth in vitro of embryoid bodies derived from teratocarcinoma.

Embryoid bodies (EB) derived from teratocarcinoma (TC) OTT6050 were cultured with ascitic liquids (AL) from animals carrying 16-, 22- and 35-day evolved EB. At the same time the presence of fibronectin (FN) in AL were analyzed by immunoblotting. Results indicate the probable existence of growth-stimulatory factors for EB, as well as the presence of FN in the 22-day AL.     

BMPs regulate differentiation of a putative visceral endoderm layer within human embryonic stem-cell-derived embryoid bodies.

Human embryonic stem cells (HESCs), pluripotent cells derived from the inner cell mass (ICM) of human blastocysts, represent a novel tool for the study of early human developmental events. When cultured in suspension with serum, HESCs form spherical structures resembling embryoid bodies (EBs). We show that differentiation of HESCs within EBs occurs radially, with central cells then undergoing apoptosis in association with EB cavitation. Cells within the outer layer of cavitating EBs display stage-specific immunoreactivity to pan-keratin, cytokeratin-8, GATA6, alpha-fetoprotein, and transthyretin specific antibodies, and hybridization to disabled-2, GATA4, and GATA6 specific riboprobes. Transmission electron microscopy of these cells reveals clathrin-coated micropinocytotic vesicles, microvilli, and many vacuoles, a phenotype consistent with mouse visceral endoderm (VE) rather than mouse definitive or parietal endoderm. When cultured in media supplemented with the BMP inhibitor noggin, or in the absence of serum, HESC derivatives do not develop the mouse VE-like phenotype. The addition of BMP-4 to noggin-treated HESCs cultured in serum or in serum-free conditions reconstituted development of the VE-like phenotype. These data demonstrate that human EBs undergo developmental events similar to those of mouse EBs and that in vitro BMP signalling induces derivatives of the human ICM to express a phenotype similar to mouse VE.     

A role for p21 (WAF1) in the cAMP-dependent differentiation of F9 teratocarcinoma cells into parietal endoderm

Combined treatment of teratocarcinoma F9 cells with retinoic acid and dibutyryl-cAMP induces the differentiation into cells with a phenotype resembling parietal endoderm. We show that the levels of cyclin-dependent kinase inhibitor p21/WAF1/Cip1 (p21) protein and mRNA are dramatically elevated at the end of this differentiation, concomitantly with the appearance of p21 in the immunoprecipitated CDK2-cyclin E complex. The induction of differentiation markers could not be achieved by expression of ectopic p21 alone and still required treatment with differentiation agents. Clones of F9 cells transfected with sense or antisense p21 cDNA constructs revealed, upon differentiation, upregulated levels of mRNA for thrombomodulin, a parietal endoderm-specific marker, or increased fraction of cells in sub-G1 phase of the cell cycle, respectively. Consistent with this observation, whereas p21 was strictly nuclear in undifferentiated cells, a large proportion of differentiated cells had p21 localized also in the cytoplasm, a site associated with the antiapoptotic function of p21. Furthermore, p21 activated the thrombomodulin promoter in transient reporter assays and the p21 mutant defective in binding to cyclin E was equally efficient in activation. The promoter activity in differentiated cells was reduced by cotransfection of p21-specific siRNA or antisense cDNA. Coexpression of p21 increased the activity of the GAL-p300(1-1303) fusion protein on the GAL sites-containing TM promoter. This implies that p21 might act through a derepression of the p300 N-terminal-residing repression domain, thereby enhancing the p300 coactivator function. As differentiation of F9 cells into parietal endoderm-like cells requires the cAMP signaling, the results together suggest that the cyclin-dependent kinase inhibitor p21 may promote specifically this pathway in F9 cells.