Disruption of the cytoskeleton-extracellular matrix linkage promotes the accumulation of plasminogen activators in F9 derived parietal endoderm

When F9 teratocarcinoma cells are treated with retinoic acid plus cyclic AMP (RACF9) they differentiate into parietal endoderm. Differentiation is accompanied by the acquisition of substrate adhesion sites and a change in the pattern of gene expression, including the synthesis of tissue-type plasminogen activator (tPA). We demonstrate here that dihydrocytochalasin B (DHCB) treatment of differentiating F9 cells prevents the assembly of a structured actin cytoskeleton and generates a more rounded and stellate cell morphology. This morphological change is accompanied by the accumulation of the usually visceral endoderm-specific marker urokinase-type plasminogen activator (uPA) and an increase in tPA levels in comparison to untreated RACF9 controls. The increase in tPA accumulation is preceded by an increase in tPA mRNA levels. These effects are reversible, with a lag, when DHCB is removed, and PA accumulation can be stimulated within 24 h when differentiated cells are exposed to DHCB. Exposure to the microtubule disrupting agent colchicine has no effect on uPA or tPA accumulation. In addition, antibody directed against the beta 1 integrin subunit can also specifically elicit increased PA production. Thus disturbing the cytoskeleton and cytoskeleton associated substrate adhesions promotes PA accumulation.     

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.     

The production of distinct forms of plasminogen activator by mouse embryonic cells

We have examined cultured parietal endoderm, visceral endoderm, and extraembryonic mesoderm cells from the mouse embryo for production of the protease plasminogen activator. All of these cell types synthesize and secrete the enzyme, but the molecular characteristics of the plasminogen activators differ as defined by the apparent molecular weight in sodium dodecyl sulfate-polyacrylamide gels, the antigenic properties as defined by two antisera to distinct plasminogen activators, and the interaction with an inhibitor present in fetal bovine serum. The parietal endoderm plasminogen activator has a predominant molecular weight of 79,000, is immunoprecipitated and inhibited by an antiserum raised against a human melanoma plasminogen activator, but not by an antiserum against mouse urokinase, and is only partially inhibited by the serum inhibitor. The visceral endoderm and extraembryonic mesoderm plasminogen activators, which are identical by all criteria, have molecular weights of 48,000, are inactivated only by the anti-urokinase antibodies, and are inhibited by an inhibitor in fetal bovine serum. These results establish the presence of at least two different forms of plasminogen activator in the early mouse embryo. The distinctive nature of the enzyme produced by parietal endoderm can be used as a diagnostic marker for this cell type at this stage of development. When F9 teratocarcinoma stem cells are induced to differentiate by retinoic acid and dibutyryl cAMP, they secrete a plasminogen activator of the parietal endoderm type.     

The role of cell interactions in the differentiation of teratocarcinoma-derived parietal and visceral endoderm

Cell interactions have been implicated in the differentiation of visceral and parietal endoderm in the developing mouse embryo. Embryoid bodies formed from F9 embryonal carcinoma cells have been useful in characterizing the events which lead to endoderm formation. As part of our effort to specify the interactions which may be involved in this process we have isolated visceral endoderm-like cells (VE) from F9 embryoid bodies and cultured them under various conditions. Using a combination of immunoprecipitation and enzyme-linked immunosorbent assay, we demonstrate that monolayer culture of these cells on a number of different substrates leads to a dramatic decrease in the level of alphafetoprotein (AFP), a VE-specific marker. Northern blot analysis of AFP mRNA indicates very low levels of this message are present after 48 hr in monolayer culture. Coincident with the drop in AFP levels is an increase in the levels of the cytokeratin Endo C and tissue plasminogen activator, both markers for parietal endoderm (PE). Morphological evidence at the ultrastructural level supports a transition from VE to PE. In contrast, the VE phenotype can be maintained in vitro by interaction with aggregates, but not monolayers, of stem cells. In addition, culturing the cells on the curved surface of gelatin-coated dextran beads, but not on a flat gelatin surface facilitates AFP expression and the cells are morphologically intermediate between VE and PE cells. The potential role of junctional complexes and cell shape are discussed.     

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.     

The Involvement of Tissue-Type Plasminogen Activator in Parietal Endoderm Outgrowth

When F9 stem cells are treated in suspension with retinoic acid, they differentiate into embryoid bodies (EBs) consisting of an inner core of undifferentiated stem cells surrounded by an outer layer of visceral endoderm (VE). When these EBs are plated onto a fibronectin (FN)-coated substrate, VE-derived parietal endoderm (PE) cells migrate onto the substrate. It has been suggested that increased levels of tPA associated with the emerging PE cells may help mediate PE outgrowth. We now show that goat anti-human tPA, an anticatalytic antibody that crossreacts with mouse tPA, and a panel of serine protease inhibitors partially inhibit PE outgrowth. Extracellular matrix (ECM) degradation analysis demonstrates that PE cell-mediated degradation of [³H]proline-labeled ECM is time- and cell concentration-dependent. A serine protease inhibitor reduced the extent of degradation, suggesting that tPA might play a role in PE outgrowth by cleaving the ECM. In support of this contention, we demonstrate that incubation of purified FN with conditioned medium plus plasminogen results in FN proteolysis. The degradation of FN is blocked by either serine protease inhibitors or goat anti-human tPA. Our data suggest that enhanced production of tPA during PE outgrowth may facilitate the migratory behavior of PE cells by mediating the degradation of ECM components such as FN.     

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.     

Evidence for the existence of an early common biochemical pathway in the differentiation of F9 cells into visceral or parietal endoderm: Modulation by cyclic AMP

The addition of dibutyryl cyclic AMP (dbcAMP) to aggregate cultures of F9 cells in medium containing retinoic acid (RA) directs the pathway of differentiation into parietal endoderm instead of visceral endoderm. We examined the levels of some of the markers that characterize the two pathways and studied the time of commitment of cells to either direction of differentiation by using immunoprecipitation and enzyme-linked immunosorbent assays (ELISA). For either pathway, the levels and patterns of laminin, type IV collagen, and fibronectin are the same on the first day of differentiation, characterized by slightly decreased levels of laminin and type IV collagen synthesis and an increased level of fibronectin synthesis. These levels reverse on the second day of culture when the pathways diverge markedly. The differentiation pathway, however, can be redirected into the alternate one; parietal endoderm cells become committed after 3 days, whereas visceral endoderm cells are able to change into parietal endoderm cells at any time. Thus, α-fetoprotein (AFP)-producing F9 embryoid bodies switched to dbcAMP-containing medium lose the capacity to synthesize AFP and start to express genes characteristic of parietal endoderm. Our results indicate that at least some visceral endoderm cells may redifferentiate into parietal endoderm cells. These phenomena thus mimic features of endoderm differentiation in the mouse embryo.     

Activators and inhibitors of the plasminogen system in Alzheimer's disease

Accumulation and deposition of Aβ is one of the main neuropathological hallmarks of Alzheimer's disease (AD) and impaired Aβ degradation may be one mechanism of accumulation. Plasmin is the key protease of the plasminogen system and can cleave Aβ. Plasmin is activated from plasminogen by tissue plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The activators are regulated by inhibitors which include plasminogen activator inhibitor-1 (PAI-1) and neuroserpin. Plasmin is also regulated by inhibitors including α2-antiplasmin and α2-macroglobulin. Here, we investigate the mRNA levels of the activators and inhibitors of the plasminogen system and the protein levels of tPA, neuroserpin and α2-antiplasmin in post-mortem AD and control brain tissue. Distribution of the activators and inhibitors in human brain sections was assessed by immunoperoxidase staining. mRNA measurements were made in 20 AD and 20 control brains by real-time PCR. In an expanded cohort of 38 AD and 38 control brains tPA, neuroserpin and α2-antiplasmin protein levels were measured by ELISA. The activators and inhibitors were present mainly in neurons and α2-antiplasmin was also associated with Aβ plaques in AD brain tissue. tPA, uPA, PAI-1 and α2-antiplasmin mRNA were all significantly increased in AD compared to controls, as were tPA and α2-antiplasmin protein, whereas neuroserpin mRNA and protein were significantly reduced. α2-macroglobulin mRNA was not significantly altered in AD. The increases in tPA, uPA, PAI-1 and α2-antiplasmin may counteract each other so that plasmin activity is not significantly altered in AD, but increased tPA may also affect synaptic plasticity, excitotoxic neuronal death and apoptosis.     

Roles for Rho/ROCK and Vinculin in Parietal Endoderm Migration

The first cell migration event in the mouse embryo is the movement of parietal endoderm cells from the surface of the inner cell mass facing the blastocoel cavity to line the inner surface of the trophectoderm. F9 embryoid bodies provide an in vitro model for this event. They have an inner core of undifferentiated stem cells surrounded by an outer visceral endoderm layer. When plated on a laminin coated substrate, visceral endoderm transitions to parietal endoderm and migrates onto the dish, away from the attached embryoid body. We now show that this outgrowth contains abundant focal complexes and focal adhesions, as well as lamellipodia and filopodia. Treatment with the ROCK inhibitor Y-27632 promotes a 2-fold increase in outgrowth, and a transition from focal adhesions and associated stress fibers, to focal complexes and a decrease in stress fibers. ROCK inhibition also leads to an increase in lamellipodia. Inhibition of RhoA by transfection of a vector encoding C3 transferase, direct administration of the C3 enzyme, or transfection of a vector encoding p190 Rho GTPase Activating Protein also promotes outgrowth and an apparent transition from focal adhesions to focal complexes. Parietal endoderm outgrowth generated using vinculin-deficient F9 stem cells migrates 2-fold further than wild type cultures, but this outgrowth retains the morphology of wild type parietal endoderm, including focal adhesions and stress fibers. Addition of Y-27632 to vinculin-null outgrowth cultures further stimulates migration an additional 2-fold, supporting the conclusion that Rho/ROCK and vinculin regulate parietal endoderm outgrowth by distinct pathways.     

Regulation of urokinase- and tissue-type plasminogen activator gene expression in the rat seminiferous epithelium

The secretion of plasminogen activator by seminiferous tubules at defined stages of the epithelial cycle is influenced both by neighboring spermatogenic cells and by hormones. We have used cRNA probes for urokinase-type (uPA) and tissue-type (tPA) plasminogen activators to analyze their mRNA levels in different stages of the epithelial cycle. Urokinase-type PA mRNA was most abundant in stages VII-VIII, while tPA mRNA levels showed smaller variations between the different stages. Both FSH and (Bu)2cAMP increased the steady-state level of tPA mRNA and tPA production without affecting those of uPA in stages VII-IX in vitro, whereas retinoic acid treatment selectively increased the concentration uPA mRNA and uPA production in stages II-VI. The results show that the expression of the uPA and tPA genes is differentially regulated in specific stages of the rat seminiferous epithelium.     

Roles for Rho/ROCK and vinculin in parietal endoderm migration

The first cell migration event in the mouse embryo is the movement of parietal endoderm cells from the surface of the inner cell mass facing the blastocoel cavity to line the inner surface of the trophectoderm. F9 embryoid bodies provide an in vitro model for this event. They have an inner core of undifferentiated stem cells surrounded by an outer visceral endoderm layer. When plated on a laminin coated substrate, visceral endoderm transitions to parietal endoderm and migrates onto the dish, away from the attached embryoid body. We now show that this outgrowth contains abundant focal complexes and focal adhesions, as well as lamellipodia and filopodia. Treatment with the ROCK inhibitor Y-27632 promotes a 2-fold increase in outgrowth, and a transition from focal adhesions and associated stress fibers, to focal complexes and a decrease in stress fibers. ROCK inhibition also leads to an increase in lamellipodia. Inhibition of RhoA by transfection of a vector encoding C3 transferase, direct administration of the C3 enzyme, or transfection of a vector encoding p190 Rho GTPase Activating Protein also promotes outgrowth and an apparent transition from focal adhesions to focal complexes. Parietal endoderm outgrowth generated using vinculin-deficient F9 stem cells migrates 2-fold further than wild type cultures, but this outgrowth retains the morphology of wild type parietal endoderm, including focal adhesions and stress fibers. Addition of Y-27632 to vinculin-null outgrowth cultures further stimulates migration an additional 2-fold, supporting the conclusion that Rho/ROCK and vinculin regulate parietal endoderm outgrowth by distinct pathways.     

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.     

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.     

Overexpression of plasminogen activators in the nucleus accumbens enhances cocaine-, amphetamine- and morphine-induced reward and behavioral sensitization

Urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) are extracellular proteases that play a role in synaptic plasticity and remodeling. Psychostimulants induce both tPA and uPA in acute and chronic drug delivery, but cocaine induces preferentially uPA, whereas morphine and amphetamine induce preferentially tPA. Specific doxycline-regulatable lentiviruses expressing these extracellular proteases have been prepared and stereotaxically injected into the nucleus accumbens. We show that tPA-overexpressing animals show greater locomotor activity and behavioral sensitization upon morphine and amphetamine treatments. These effects could be fully suppressed by doxycycline or when tPA had been silenced using small interfering RNAs (siRNAs)-expressing lentiviruses. Furthermore, animals infected with lentiviruses expressing uPA show enhanced conditional place preference for cocaine compared with tPA-overexpressing animals. In contrast, tPA-overexpressing animals when administered amphetamine or morphine showed greater place preference compared with uPA-overexpressing animals. The effects are suppressed when tPA has been silenced using specific siRNAs-expressing vectors. Tissue-type plasminogen activator and uPA possibly induce distinct behaviors, which may be interpreted according to their differential pattern of activation and downstream targets. Taken together, these data add further evidence for a significant function of extracellular proteases tPA and uPA in addiction and suggest a differential role of plasminogen activators in this context.     

Insulin-like growth factor-binding protein-5 activates plasminogen by interaction with tissue plasminogen activator, independently of its ability to bind to plasminogen activator inhibitor-1, insulin-like growth factor-I, or heparin

Transgenic mice expressing IGFBP-5 in the mammary gland exhibit increased cell death and plasmin generation. Because IGFBP-5 has been reported to bind to plasminogen activator inhibitor-1 (PAI-1), we determined the effects of this interaction in HC11 cells. PAI-1 prevented plasmin generation from plasminogen and inhibited cleavage of focal adhesions, expression of caspase 3, and cell death. IGFBP-5 could in turn prevent the effects of PAI-1. IGFBP-5 mutants with reduced affinity for IGF-I (N-term) or deficient in heparin binding (HEP- and C-term E and F) were also effective. This was surprising because IGFBP-5 reportedly interacts with PAI-1 via its heparin-binding domain. Biosensor analysis confirmed that, although wild-type IGFBP-5 and N-term both bound to PAI-1, the C-term E had greatly decreased interaction with PAI-1. This suggests that IGFBP-5 does not antagonize the actions of PAI-1 by a direct molecular interaction. In a cell-free system, using tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) to activate plasminogen, PAI-1 inhibited plasmin generation induced by both activators, whereas IGFBP-5 prevented the effects of PAI-1 on tPA but not uPA. Furthermore, we noted that IGFBP-5 activated plasminogen to a greater extent than could be explained solely by inhibition of PAI-1, suggesting that IGFBP-5 could directly activate tPA. Indeed, IGFBP-5 and the C-term E and F were all able to enhance the activity of tPA but not uPA. These data demonstrate that IGFBP-5 can enhance the activity of tPA and that this can result in cell death induced by cleavage of focal adhesions. Thus IGFBP-5 can induce cell death by both sequestering IGF-I and enhancing plasmin generation.     

Identification of murine extra-embryonic endodermal cells by reaction with teratocarcinoma basement membrane antiserum

Embryonal carcinoma cells from the PSA1 cell line will differentiate in vitro to form structures called embryoid bodies composed of an inner core of embryonal carcinoma cells surrounded by a basement membrane matrix and an outer layer of extra-embryonic endodermal cells. Immunization of rabbits with basement membranes isolated from embryoid bodies resulted in an antiserum, which binds to fixed extra-embryonic endodermal cells of either embryonic or teratocarcinoma origin but does not bind substantially to mouse embryonal carcinoma cells, fibroblasts, myoblasts or erythroleukemic cells. The F9-22 embryonal carcinoma cell line normally differentiates only to a very limited extent in vitro or in vivo. However, incubation of these cells in medium containing retinoic acid results in the appearance of cells resembling extra-embryonic endoderm. The embryoid body basement membrane antibodies were used to measure, by flow microfluorometry, the appearance of reactive cells in F9-22 cultures treated with retinoic acid. The kinetics of appearance of cells reactive with the basement membrane antibodies are similar to the kinetics of appearance of cells secreting plasminogen activator, a known marker of extraembryonic endoderm.     

Parietal endoderm secreted SPARC promotes early cardiomyogenesis in vitro

Cardiomyogenesis proceeds in the presence of signals emanating from extra-embryonic lineages emerging before and during early eutherian gastrulation. In embryonic stem cell derived embryoid bodies, primitive endoderm gives rise to visceral and parietal endoderm. Parietal endoderm undergoes an epithelial to mesenchymal transition shortly before first cardiomyocytes start to contract rhythmically. Here, we demonstrate that Secreted Protein, Acidic, Rich in Cysteine, SPARC, predominantly secreted by mesenchymal parietal endoderm specifically promotes early myocardial cell differentiation in embryoid bodies. SPARC enhanced the expression of bmp2 and nkx2.5 in embryoid bodies and fetal cardiomyocytes. Inhibition of either SPARC or Bmp2 attenuated in both cases cardiomyogenesis and downregulated nkx2.5 expression. Thus, SPARC directly affects cardiomyogenesis, modulates Bmp2 signaling, and contributes to a positive autoregulatory loop of Bmp2 and Nkx2.5 in cardiomyocytes.     

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.