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.     

Cell autonomous sorting and surface positioning in the formation of primitive endoderm in embryoid bodies

The differentiation and formation of the primitive endoderm in early embryos can be mimicked in vitro by the aggregation of embryonic stem cells to form embryoid bodies. We present morphological evidence that primitive endoderm cells often first locate in the interior of embryoid bodies and subsequently migrate to the surface. Cell mixing experiments indicate that surface positioning is an intrinsic property of endoderm epithelial cells. Moreover, Disabled-2 (Dab2) is required for surface sorting and positioning of the endoderm cells: when Dab2 expression was eliminated, the differentiated endoderm epithelial cells distributed throughout the interior of the embryoid bodies. Surprisingly, E-cadherin is dispensable for primitive endoderm differentiation and surface sorting in embryoid bodies. These results support the model that primitive endoderm cells first emerge in the interior of the inner cell mass and are subsequently sorted to the surface to form the primitive endoderm.     

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.     

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.     

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.     

Identification of small signalling molecules promoting cardiac-specific differentiation of mouse embryonic stem cells.

Identification of signalling cascades involved in cardiomyogenesis is crucial for optimising the generation of cardiomyocytes from embryonic stem cells (ES cells) (in vitro). We used a transgenic ES cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of the alpha-myosin heavy chain (alpha-MHC) promoter (palphaMHC-EGFP) to investigate the effects of 33 small molecules interfering with several signalling cascades on cardiomyogenesis. Interestingly, the L-Type Ca2+ channel blocker Verapamil as well as Cyclosporin, an inhibitor of the protein phosphatase 2B, exerted the most striking pro-cardiomyogenic effect. Forskolin (adenylate cyclase stimulator) exerted the most striking anti-cardiomyogenic effect. The cardiomyogenic effect of Cyclosporin and Verapamil correlated with an expression of early cardiac markers Nkx2.5 and GATA4.Compared to the effects on late developmental stage embryoid bodies (EBs) stimulation of early developmental stage EBs (1-day old) with Verapamil or Cyclosporin for 48 h resulted in a potent cardiomyogenic effect. Accordingly, enhanced expression of alpha-MHC mRNA and EGFP mRNA was observed after stimulation of the early developmental stage EBs for 48 h. No expression of alpha-smooth muscle actin or platelet endothelial cell adhesion molecule-1 (PECM-1) as well as of neuronal genes (Nestin, Neurofilament H) has been observed demonstrating a preferentially pro-cardiomyogenic effect by both molecules.     

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.     

Plasminogen activator expression in F9 teratocarcinoma embryoid bodies and their endoderm derivatives

Plasminogen activators are believed to play an important role in tissue remodeling and cell migration. During mouse embryogenesis, visceral endoderm secretes urokinase-type plasminogen activator (uPA) whereas parietal endoderm secretes tissue-type plasminogen activator (tPA). Visceral endoderm from F9 embryoid bodies can transdifferentiate into parietal endoderm under the appropriate culture conditions. We have examined at the protein and mRNA levels the type of plasminogen activator expressed in whole embryoid bodies, visceral endoderm and its parietal endoderm derivatives. Our experiments show that the visceral endoderm on F9 embryoid bodies synthesizes and secretes substantial amounts of both tPA and uPA. In contrast, the parietal endoderm derived directly from the visceral endoderm secretes dramatically increased levels of tPA and decreases production of uPA to low or below detectable levels. These data support the finding that visceral endoderm can transdifferentiate to parietal endoderm. In addition, this transition provides an excellent model for studying the molecular basis of the coincident down- and upregulation of the two plasminogen activators as well as their potential function during embryogenesis.     

Nodal inhibits differentiation of human embryonic stem cells along the neuroectodermal default pathway

Genetic studies in fish, amphibia, and mice have shown that deficiency of Nodal signaling blocks differentiation into mesoderm and endoderm. Thus, Nodal is considered as a major inducer of mesendoderm during gastrulation. On this basis, Nodal is a candidate for controlling differentiation of pluripotent human embryonic stem cells (hESCs) into tissue lineages with potential clinical value. We have investigated the effect of Nodal, both as a recombinant protein and as a constitutively expressed transgene, on differentiation of hESCs. When control hESCs were grown in chemically defined medium, their expression of markers of pluripotency progressively decreased, while expression of neuroectoderm markers was strongly upregulated, thus revealing a neuroectodermal default mechanism for differentiation in this system. hESCs cultured in recombinant Nodal, by contrast, showed prolonged expression of pluripotency marker genes and reduced induction of neuroectoderm markers. These Nodal effects were accentuated in hESCs expressing a Nodal transgene, with striking morphogenetic consequences. Nodal-expressing hESCs developing as embryoid bodies contained an outer layer of visceral endoderm-like cells surrounding an inner layer of epiblast-like cells, each layer having distinct gene expression patterns. Markers of neuroectoderm were not upregulated during development of Nodal-expressing embryoid bodies, nor was there induction of markers for definitive mesoderm or endoderm differentiation. Moreover, the inner layer expressed markers of pluripotency, characteristic of undifferentiated hESCs and of epiblast in mouse embryos. These results could be accounted for by an inhibitory effect of Nodal-induced visceral endoderm on pluripotent cell differentiation into mesoderm and endoderm, with a concomitant inhibition of neuroectoderm differentiation by Nodal itself. There could also be a direct effect of Nodal in the maintenance of pluripotency. In summary, analysis of the Nodal-expressing phenotype suggests a function for the transforming growth factor-beta (TGF-beta) growth factor superfamily in pluripotency and in early cell fate decisions leading to primary tissue layers during in vitro development of pluripotent human stem cells. The effects of Nodal on early differentiation illustrate how hESCs can augment mouse embryos as a model for analyzing mechanisms of early mammalian development.     

Formation of a Polarised Primitive Endoderm Layer in Embryoid Bodies Requires Fgfr/Erk Signalling

The primitive endoderm arises from the inner cell mass during mammalian pre-implantation development. It faces the blastocoel cavity and later gives rise to the extraembryonic parietal and visceral endoderm. Here, we investigate a key step in primitive endoderm development, the acquisition of apico-basolateral polarity and epithelial characteristics by the non-epithelial inner cell mass cells. Embryoid bodies, formed from mouse embryonic stem cells, were used as a model to study this transition. The outer cells of these embryoid bodies were found to gradually acquire the hallmarks of polarised epithelial cells and express markers of primitive endoderm cell fate. Fgf receptor/Erk signalling is known to be required for specification of the primitive endoderm, but its role in polarisation of this tissue is less well understood. To investigate the function of this pathway in the primitive endoderm, embryoid bodies were cultured in the presence of a small molecule inhibitor of Mek. This inhibitor caused a loss of expression of markers of primitive endoderm cell fate and maintenance of the pluripotency marker Nanog. In addition, a mislocalisation of apico-basolateral markers and disruption of the epithelial barrier, which normally blocks free diffusion across the epithelial cell layer, occurred. Two inhibitors of the Fgf receptor elicited similar phenotypes, suggesting that Fgf receptor signalling promotes Erk-mediated polarisation. This data shows that primitive endoderm cells of the outer layer of embryoid bodies gradually polarise, and formation of a polarised primitive endoderm layer requires the Fgf receptor/Erk signalling pathway.     

Visceral endoderm induces specification of cardiomyocytes in mice

The endoderm plays an inductive role in the formation of cardiomyocytes in many vertebrates. Here, we provide further evidence for this in the mouse and demonstrate enhanced cardiomyogenesis in mouse embryonic stem cells cultured in the presence of native visceral endoderm. Isolated mesoderm from late-primitive streak stage mouse embryos that still have an open proamniotic canal had a reduced capacity to form cardiomyocytes after 4 days in culture compared with mesoderm isolated from later stages but prior to cardiomyogenesis. Moreover, removal of the visceral endoderm but not the primitive streak reduced the formation of beating areas in embryo explants in culture. Coculture with the END2 cell line, which has visceral endoderm-like properties, restored the formation of beating areas. Immunohistochemical analysis showed that the expected candidate signaling pathways downstream of Wnts and bone morphogenetic proteins (BMPs) were active in the embryo at the appropriate time and place to be involved. Overall, the results show that, as in other vertebrates, the (visceral) endoderm plays an important role in the early events of mouse cardiomyogenesis.     

SPARC synthesis in pre-implantation and early post-implantation mouse embryos

Summary SPARC (secreted protein acidic and rich in cysteine), also known as osteonectin and BM-40, is a secreted protein associated with a variety of embryonic and adult tissue and cell types, including placenta, parietal and visceral endoderm, certain epithelia (e.g. gut, skin, glandular epithelia), and regions of active chondrogenesis and osteogenesis. Although much is known concerning the tissue distribution of this protein, neither the time and location of its initial appearance nor its functions during embryogenesis have been clearly established. We identified the location of SPARC on two-dimensional protein gels. By using two-dimensional gel analysis of both pre- and post-implantation stage mouse embryos, we find that SPARC is initially synthesized between 3.5 and 4.5 days of embryogenesis. This is the earliest time during development at which synthesis of SPARC has been demonstrated. Inner cell masses isolated from 4.5 day blastocysts synthesize SPARC indicating that either primitive ectoderm, primitive endoderm, or both produce this protein. SPARC synthesis is also detectable in isolated trophoblast vesicles. Thus, SPARC is synthesized not only in placenta, parietal endoderm, and visceral endoderm, but in the precursors of these tissues as well. Examination of 7.5 day embryos reveals that SPARC is synthesized in isolated parietal yolk sac and in whole extraembryonic and embryonic regions. Relative to other proteins, synthesis of SPARC was most prevalent in the parietal yolk sac. The possible implications of SPARC synthesis as early as 4.5 days are discussed.     

Migration of F9 parietal endoderm cells is regulated by the ERK pathway

Cell migration is regulated by the action of many signaling pathways that are activated in specific regions of migrating cells. Extracellular regulated kinase 1/2 (ERK) signaling can modulate the migration of cells by controlling the turnover of focal adhesions and the dynamics of actin polymerization. Focal adhesion turnover is necessary for cell migration, and the formation of strong actin stress fibers and mature focal adhesions puts the brakes on cell migration. We used F9 wild-type and vinculin null (vin-/-) parietal endoderm (PE) outgrowth to study the role of the ERK signaling pathway in cell migration. Upon plating of F9 embryoid bodies (EBs) onto laminin-coated dishes, PE cells migrate away from the EBs, providing an in vitro model for studying directed migration of this embryonic cell type. Our results suggest that the ERK pathway regulates PE cell migration by affecting the formation of focal adhesions and lamellipodia through the action of myosin light chain kinase (MLCK).     

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.