Interactive Fly, Drosophila

Role of FGF during gastrulation (part 2/2)

Analyses using amphibian embryos have proposed that induction and anteroposterior patterning of the central nervous system is initiated by signals that are produced by the organizer and organizer-derived axial mesoderm. However, here it is shown that the initial anteroposterior pattern of the zebrafish central nervous system depends on the differential competence of the epiblast and is not imposed by organizer-derived signals. This anteroposterior information is present throughout the epiblast in ectodermal cells that normally give rise both to neural and non-neural derivatives. Because of this information, organizer tissues transplanted to the ventral side of the embryo induce neural tissue but the anteroposterior identity of the induced neural tissue is dependent on the position of the induced tissue within the epiblast. Thus, otx2, an anterior neural marker, was only induced in the anterior regions of the embryo, irrespective of the position of the grafts. Similarly, hoxa-1, a posterior neural marker is induced only in the posterior regions. The boundary of each ectopic expression domain on the ventral side is always at an equivalent latitude to that of the endogenous expression of the dorsal side of the embryo. The anteroposterior specification of the epiblast is independent of the dorsoventral specification of the embryo because neural tissues induced in the ventralized embryos also show anteroposterior polarity. Cell transplantation and RNA injection experiments show that non-axial marginal mesoderm and FGF signaling is required for anteroposterior specification of the epiblast. However, the requirement for FGF signaling is indirect because cells with compromised ability to respond to FGF can still respond to anteroposterior positional information (Koshida, 1998).

A sensitive assay for MAP kinase (See Drosophila Rolled) activity was used to investigate the role of endogenous fibroblast growth factor (FGF)-activated MAP kinase in early Xenopus embryonic patterning. MAP kinase activity is low during cleavage stages and increases significantly during gastrulation. The temporal profile of this activity correlates well with the expression pattern of Xenopus eFGF. Spatially, MAP kinase activity is lowest in animal pole tissue and higher in vegetal pole cells and the marginal zone. Endogenous MAP kinase activity is FGF receptor-dependent, demonstrating that FGF signaling is active in all three germ layers of the early embryo. This activity is necessary for normal expression of Mix.1, a mesoendodermal marker, in the endoderm as well as in the mesoderm, indicating that MAP kinase plays a functional role in patterning both of these germ layers. Spatial and temporal changes in MAP kinase activation during gastrulation also suggest a role for FGF signaling in this process. Embryonic wounding during dissection results in significant stimulation of this pathway, providing a possible explanation for earlier observations of effects of surgical manipulation on cell fate in early embryos (LaBonne, 1997).

The mesoderm of Xenopus laevis arises through an inductive interaction in which signals from the vegetal hemisphere of the embryo act on overlying equatorial cells. One candidate for an endogenous mesoderm-inducing factor is activin, a member of the TGFbeta superfamily. Activin is of particular interest because it induces different mesodermal cell types in a concentration-dependent manner, suggesting that it acts as a morphogen. Low doses of activin do not induce expression; intermediate concentrations induce high levels, and still higher concentrations suppress expression. These concentration-dependent effects are exemplified by the response of Xbra, whose expression is induced in ectodermal tissue by low concentrations of activin, but not by high concentrations. Xbra therefore offers an excellent paradigm for studying the way in which a morphogen gradient is interpreted in vertebrate embryos. An examination was carried out of the trancriptional regulation of Xbra2, a pseudoallele of Xbra that shows an identical response to activin. Sequences of 381bp, 5' to the Xbra2 transcription start site, are sufficient to confer responsiveness both to FGF and, in a concentration-dependent manner, to activin. The suppression of Xbra expression at high concentrations of activin is mediated by paired-type homeobox genes such as goosecoid, Mix.1, and Xotx2. Mutation of Hox binding sites in the region -174/-152bp the start site abolishes suppression of activin-induced activity by goosecoid (Latinkic, 1997).

XLPOU91, a POU-homeobox gene is expressed in a narrow window during early Xenopus development. XLPOU91 is most similar to the mammalian class V POU-domain gene Oct3/4 (mammalian class V genes have no known Drosophila homolog). Like XLPOU91, Oct3/4 is expressed in early embryonic cells; its expression is downregulated in the embryo at about the time of gastrulation. Ectopic expression of XLPOU91 RNA causes severe posterior truncations in embryos without inhibiting the formation of Spemann's organizer. Ectopic XLPOU91 expression also inhibits mesoderm induction by fibroblast growth factor (FGF) and activin in animal cap explants. Using antisense RNA, endogenous XLPOU91 protein is depleted in animal caps. Gastrula-stage animal caps expressing XLPOU91 antisense RNA do not lose competence for response to FGF; unlike controls, these animal caps express XBra after FGF treatment. Endogenous XLPOU91 levels are peaking when FGF mesoderm-inducing competence is lost in animal caps. Thus XLPOU91 protein may act as a competence switch during early development, as XLPOU91 levels increase in the embryo, the mesoderm response to FGF is lost. It is suggested that XLPOU91 acts as an endogenous competence regulator. Classic embryological experiments have shown that ectoderm loses its ability to respond to mesoderm induction during gastrulation. This occurs through a built-in timing mechanism in a cell autonomous manner. Increasing or decreasing XLPOU91 levels may determine whether a cell can respond to FGF and make mesoderm at a given time during development. Thus, XLPOU91, which increases to peak levels at gastrula stages, could act as a switch to modify FGF mesoderm-response in the embryo (Henig, 1998).

An in vitro differentiation assay has been developed to characterize the ability of peptide growth factors to induce differentiation in mouse epiblast. Culturing explants of mouse anterior epiblast (a tissue normally fated to give rise to neuroectoderm and surface ectoderm) in a serum-free, chemically defined medium with 10-50 ng/ml of FGF-2, induces gross changes in cell morphology. Treated cells adopt an elongated, flattened morphology but do not migrate from the explant. Instead, FGF-2-treated cells condense into multicellular mounds or ridges. Immunocytochemistry shows that cells in treated explants express vimentin; in situ hybridization demonstrates that FGF-2 induces the expression of brachyury, goosecoid, and myo-D in regions of treated explants displaying morphological differentiation. Control explants cultured with platelet-derived growth factor AA (PDGF AA), transforming growth factor-beta 1 (TGF-beta 1), or in defined medium alone show no morphological or biochemical differentiation. These results indicate that in vitro, FGF-2 alters the fate of mouse anterior epiblast from ectoderm to mesoderm. Cell migration, which is characteristic of primitive streak mesoderm in vivo, is not induced by FGF-2 in these assays. However, the changes in morphology and the expression of mesodermal genes in vitro do support an early role for FGF signaling in the induction of mouse primitive streak mesoderm, as well as in later patterning events during embryogenesis (Burdsal, 1998).

FGF-4 appears to be the first FGF expressed during mammalian development. Its expression has been observed at the RNA level as early as the four-cell stage. By the blastocyst stage, FGF-4 mRNA expression is restricted to the cells of the inner cell mass, which give rise to the three embryonic germ layers. As the embryo undergoes gastrulation, FGF-4 mRNA is restricted to the primitive streak, where it continues to be expressed for several additional days. After organogenesis is initiated, FGF-4 is expressed in many tissues, including the myotome cells of mature somites, the branchial arches, and the apical ectodermal ridge of the developing limb. Mouse embryos with both FGF-4 alleles inactivated are developmentally arrested shortly after implantation. To understand the roles of FGF-4 during early development, genetically engineered embryonic stem (ES) cells were prepared that were unable to produce FGF-4. This paper describes the isolation and characterization of ES cells with both FGF-4 alleles inactivated. The FGF-4-/- ES cells do not require FGF-4 to proliferate in vitro, and addition of FGF-4 to the medium has little or no effect on their growth. Thus, FGF-4 does not appear to act as an autocrine growth factor for cultured ES cells. FGF-4-/- ES cells, like their unmodified counterparts, are capable of forming highly complex tumors in syngeneic mice composed of a wide range of differentiated cells types, including neural tissue, glandular epithelium, and muscle. In addition, the FGF-4-/- ES cells can differentiate in vitro after exposure to retinoic acid; however, the growth and/or survival of the differentiated cells is severely compromised. Importantly, addition of FGF-4 to the culture medium dramatically increases the number of differentiated cells derived from the FGF-4-/- ES cells, in particular cells with many of the properties of parietal extraembryonic endoderm. There are differences in the RNA profiles expressed by the differentiated progeny formed in vitro from FGF-4-/- ES cells and FGF-4+/+ ES cells when they are cultured with FGF-4. It is concluded that certain lineages formed in vitro are affected by the inactivation of the FGF-4 gene, in particular specific cells that form during the initial stage of ES cell differentiation. Thus, ES cells with both FGF-4 alleles inactivated should shed light on the important roles of FGF-4 during the early stages of mammalian development and help determine why FGF-4-/- embryos die shortly after implantation (Wilder, 1997).

The major mesodermal tissues of ascidian larvae are muscle, notochord and mesenchyme. They are derived from the marginal zone surrounding the endoderm area in the vegetal hemisphere. Muscle fate is specified by localized ooplasmic determinants, whereas specification of notochord and mesenchyme requires inducing signals from endoderm at the 32-cell stage. All endoderm precursors are able to induce formation of notochord and mesenchyme cells in presumptive notochord and mesenchyme blastomeres, respectively, indicating that the type of tissue induced depends on differences in the responsiveness of the signal-receiving blastomeres. Basic fibroblast growth factor (bFGF), but not activin A, induces formation of mesenchyme cells as well as notochord cells. Treatment of mesenchyme-muscle precursors isolated from early 32-cell embryos with bFGF promotes mesenchyme fate and suppresses muscle fate, which is a default fate assigned by the posterior-vegetal cytoplasm (PVC) of the eggs. The sensitivity of the mesenchyme precursors to bFGF reaches a maximum at the 32-cell stage, and the time required for effective induction of mesenchyme cells is only 10 minutes, features similar to those of notochord induction. These results support the idea that the distinct tissue types, notochord and mesenchyme, are induced by the same signaling molecule originating from endoderm precursors. The PVC causes the difference in the responsiveness of notochord and mesenchyme precursor blastomeres. Removal of the PVC results in loss of mesenchyme and in ectopic notochord formation. In contrast, transplantation of the PVC leads to ectopic formation of mesenchyme cells and loss of notochord. Thus, in normal development, notochord is induced by an FGF-like signal in the anterior margin of the vegetal hemisphere, where PVC is absent, and mesenchyme is induced by an FGF-like signal in the posterior margin, where PVC is present (Kim, 2000).

In vertebrate embryos, the precursor cells of the central nervous system (CNS) are induced by signaling from the organizer region. A novel vertebrate achaete-scute homolog, cash4, is expressed in the presumptive posterior nervous system in response to such signaling. cash4 is first expressed in epiblast cells flanking the late-phase organizer (Hensen's node). cash4 is induced by Hensen's node as evidenced by the ability of Hensen's node cells to induce cash4 when grafted into the presumptive anterior neural plate, a region that does not normally generate cells that express cash4. These node-derived signals can be mimicked in vivo by the activity of fibroblast growth factor (FGF), suggesting that a member of the FGF family of signaling molecues is involved in the activation of cash4 expression in the presumptive posterior CNS. cash4 functions as a proneural gene downstream of node-derived signals (including FGF) to promote the formation of the neural precursors that will give rise to the posterior CNS in the chick embryo (Henrique, 1997).

The polymerase chain reaction (PCR)-based differential display methodology was used to identify a transcript whose expression levels increase in Xenopus embryo explants during mesoderm induction by fibroblast growth factor. The transcript, named er1 (early response 1), is coded for by a gene with a single open reading frame predicted to encode a protein of 493 amino acid residues. The ER1 amino acid contains three regions of similarity to the rat and human proteins encoded by the metastasis-associated gene, mta1, and two regions of similarity to a Caenorhabditis elegans sequence which is also similar to mta1. The fibroblast growth factor-induced increase in er1 steady-state levels is not dependent on de novo protein synthesis, demonstrating that er1 is an immediate-early gene. A single 2.8-kilobase pair mRNA was observed, predominantly during the initial cleavage and blastula stages of Xenopus development, with little or no detectable mRNA during subsequent development. Er1 levels peak during late blastula. There are two putative nuclear localization signals, four highly acidic regions clustered at the N terminus, and a proline-rich region located near the C terminus. The ER1 protein is targeted exclusively to the nucleus. The N-terminal acidic region is a potent transactivator. These data suggest that ER1 may function as a transcription factor (Paterno, 1997).

Patterning along the anteroposterior (A/P) axis involves the interplay of secreted and transcription factors that specify cell fates in the mesoderm and neuroectoderm. While FGF and homeodomain proteins have been shown to play different roles in posterior specification, the network coordinating their effects remains elusive. The function of Gli zinc-finger proteins in mesodermal A/P patterning has been examined. Gli2 is sufficient to induce ventroposterior development, functioning in the FGF-brachyury regulatory loop. Gli2 directly induces brachyury, a gene required and sufficient for mesodermal development, and Gli2 is in turn induced by FGF signaling. Moreover, the homeobox gene Xhox3, a critical determinant of posterior development, is also directly regulated by Gli2. Gli3, but not Gli1, has an activity similar to that of Gli2 and is expressed in ventroposterior mesoderm after Gli2. These findings uncover a novel function of Gli proteins, previously only known to mediate hedgehog signals, in the maintenance and patterning of the embryonic mesoderm. More generally, these results suggest a molecular basis for an integration of FGF and hedgehog inputs in Gli-expressing cells that respond to these signals (Brewster, 2000).

Previous work has shown that Gli2 can be induced by SHH signaling in frog embryos, and that it can mediate some of the effects of SHH. FGF also induces Gli2, although it remains unclear which factors directly initiate its expression in mesoderm, as this is difficult to separate from the general induction of mesoderm by FGF or TGFbeta family signals. While Gli2/3 function in mesoderm may have nothing to do with HH signaling, HH genes have been reported to be expressed at low levels throughout the gastrula marginal zone, raising the possibility that Gli2 and Gli3 activity in mesoderm could be responsive to HH signals by analogy with some of its later roles in neural development. For example, a low tonic HH signal throughout the marginal zone could attenuate the formation of putative Gli3 repressors, a process regulated by the SHH signaling pathway, thus allowing Gli2 and Gli3 activator forms to function in ventroposterior development. The fact that misexpression of HHs at early stages has no obvious consequence on mesodermal development could be consistent with this possibility if repressor forms were not required in mesoderm. In mice, loss of SHH, Gli2 or Gli3 function does not appear to affect the early embryonic mesoderm, possibly indicating that Gli proteins could have partially divergent roles in different organisms (Brewster, 2000).

The role of Gli2 in FGF signaling, the ability of FGF and SHH to induce its expression and its partial mediation of SHH functions suggest a mechanism for a possible integration of FGF and HH signaling in tissues in which these signals act on the same Gli-expressing cells. SHH can act through Gli1, and Gli3 has an antagonistic relationship with SHH/Gli1. In contrast, SHH can also act through Gli2 in some contexts, but in others, Gli2 can instead antagonize the actions of SHH and Gli1. A context-dependent function of Gli2 could therefore underlie the sometimes synergistic and sometimes antagonistic effects of FGFs and HHs. Similarly, antagonism between HH and FGF signaling could result from their use of Gli1 and Gli3, respectively. This model may be particularly relevant for Gli-expressing precursor cells. For example, SHH is a known mitogen for cerebellar granule precursors and FGF can partially inhibit this effect. Because Wnt signaling has been recently suggested to affect Gli2 and Gli3 expression in chick somites, a challenge of ongoing studies is to elucidate how different signaling inputs regulate Gli function in vertebrate development and disease (Brewster, 2000).

In early Ciona savignyi embryos, nuclear localization of ß-catenin is the first step of endodermal cell specification, and triggers the activation of various target genes. A cDNA for Cs-FGF4/6/9, a gene activated downstream of ß-catenin signaling, was isolated and shown to encode an FGF protein with features of both FGF4/6 and FGF9/20. The early embryonic expression of Cs-FGF4/6/9 is transient and the transcript is seen in endodermal cells at the 16- and 32-cell stages, in notochord and muscle cells at the 64-cell stage, and in nerve cord and muscle cells at the 110-cell stage; the gene is then expressed again in cells of the nervous system after neurulation. When the gene function is suppressed with a specific antisense morpholino oligo, the differentiation of mesenchyme cells is completely blocked, and the fate of presumptive mesenchyme cells appears to change into that of muscle cells. The inhibition of mesenchyme differentiation is abrogated by coinjection of the morpholino oligo and synthetic Cs-FGF4/6/9 mRNA. Downregulation of ß-catenin nuclear localization results in the absence of mesenchyme cell differentiation due to failure of the formation of signal-producing endodermal cells. Injection of synthetic Cs-FGF4/6/9 mRNA in ß-catenin-downregulated embryos evokes mesenchyme cell differentiation. These results strongly suggest that Cs-FGF4/6/9 produced by endodermal cells acts an inductive signal for the differentiation of mesenchyme cells. In contrast, the role of Cs-FGF4/6/9 in the induction of notochord cells is partial; the initial process of the induction is inhibited by Cs-FGF4/6/9 morpholino oligo, but notochord-specific genes are expressed later to form a partial notochord. Thus, Ciona FGF4/6/9 is a target of ß-catenin signaling, is expressed transiently in endodermal cells of early embryos, and functions as an inductive signal for the differentiation of mesenchyme cells (Imai, 2002a).

A key issue for understanding the early development of the chordate body plan is how the endoderm induces notochord formation. In the ascidian Ciona, nuclear accumulation of ß-catenin is the first step in the process of endoderm specification. Nuclear accumulation of ß-catenin directly activates the gene (Cs-FoxD) for a winged helix/forkhead transcription factor and this gene is expressed transiently at the 16- and 32-cell stages in endodermal cells. The function of Cs-FoxD, however, is not associated with differentiation of the endoderm itself but is essential for notochord differentiation or induction. In addition, it is likely that the inductive signal that appears to act downstream of Cs-FoxD does not act over a long range. It has been suggested that FGF or Notch signal transduction pathway mediates ascidian notochord induction. Previous work suggests that Cs-FGF4/6/9 is partially involved in the notochord induction. The present experimental results suggest that the expression and function of Cs-FGF4/6/9 and Cs-FoxD are not interdependent, and that the Notch pathway is involved in B-line notochord induction (B-line cells represent a secondary notochord lineage) downstream of Cs-FoxD (Imai, 2002b).

Specification of germ layers is a crucial event in early embryogenesis. In embryos of the ascidian, Halocynthia roretzi, endoderm cells originate from two distinct lineages in the vegetal hemisphere. Cell dissociation experiments suggest that cell interactions are required for posterior endoderm formation, which has hitherto been thought to be solely regulated by localized egg cytoplasmic factors. Without cell interaction, every descendant of posterior-vegetal blastomeres, including endoderm precursors, assumes muscle fate. Cell interactions are required for suppression of muscle fate and thereby promote endoderm differentiation in the posterior endoderm precursors. The cell interactions take place at the 16- to 32-cell stage. Inhibition of cell signaling by FGF receptor and MEK inhibitor also support the requirement of cell interactions. Consistently, FGF was a potent signaling molecule, whose signaling is transduced by MEK-MAPK. By contrast, such cell interactions are not required for formation of the anterior endoderm. Another redundant signaling molecule, most likely BMP, is be involved in the posterior endoderm formation. Suppression of the function of macho-1, an odd-paired-related muscle determinant in ascidian eggs, by antisense oligonucleotide is enough to allow autonomous endoderm specification. Therefore, the cell interactions induce endoderm formation by suppressing the function of macho-1, which is to promote muscle fate. These findings suggest the presence of novel mechanisms that suppress functions of inappropriately distributed maternal determinants via cell interactions after embryogenesis starts. Such cell interactions would restrict the regions where maternal determinants work, and play a key role in marking precise boundaries between precursor cells of different tissue types (Kondoh, 2003).

Studies in fish and amphibia have shown that graded Bmp signalling activity regulates dorsal-to-ventral (DV) patterning of the gastrula embryo. In the ectoderm, it is thought that high levels of Bmp activity promote epidermal development ventrally, whereas secreted Bmp antagonists emanating from the organiser induce neural tissue dorsally. However, in zebrafish embryos, the domain of cells destined to contribute to the spinal cord extends all the way to the ventral side of the gastrula, a long way from the organiser. In vegetal (trunk and tail) regions of the zebrafish gastrula, neural specification is initiated at all DV positions of the ectoderm in a manner that is unaffected by levels of Bmp activity and independent of organiser-derived signals. Instead, Fgf activity is required to induce vegetal prospective neural markers and can do so without suppressing Bmp activity. Bmp signalling is shown to occur within the vegetal prospective neural domain and Bmp activity promotes the adoption of caudal fate by this tissue (Kudoh, 2004).

To explore the epistatic relationships between the Fgf and Bmp pathways, the consequences of locally activating or suppressing Fgf signalling were examined. fgf3-expressing ectodermal cells transplanted into animal pole regions of host embryos induce sox3 non-autonomously in surrounding host cells. This induction still occurs if the donor cells are from embryos co-expressing a truncated Fgf receptor, suggesting that the Fgf signal from the donor cells acts directly on the host. When both donor and host cells are overexpressing bmp2b, fgf3-expressing cells still induce sox3 and suppress expression of the epidermal marker gene, foxi1, suggesting that exogenous Bmp activity does not block induction of prospective neural marker genes by Fgf (Kudoh, 2004).

Next, Fgf signalling was locally suppressed by transplanting truncated Fgf receptor (XFD) expressing donor cells to various positions in the prospective neural ectoderm of host embryos. sox3 expression was suppressed in XFD-expressing cells transplanted to dorsal, lateral or ventral vegetal ectoderm, and foxi1 was ectopically induced in transplants targeted to ventral-vegetal ectoderm. These results suggest that ventral vegetal ectoderm needs to receive Fgf to express sox3, otherwise it expresses the prospective epidermal marker, foxi1 (Kudoh, 2004).

To directly assess if Fgf signals are essential for vegetal ectoderm to form neural tissue, the eventual fate was traced of XFD-expressing donor cells transplanted to wild-type hosts. In these experiments, labelled wild-type cells were co-transplanted with XFD-expressing cells to the same locations in the vegetal ectoderm of unlabelled host embryos at the end of blastula stage. When the donor cells were transplanted to the dorsal side, wild-type cells primarily contributed to the hindbrain, whereas the XFD-expressing cells localised more anteriorly, mainly in the midbrain. However, when transplanted to ventral vegetal ectoderm, wild-type donor cells contributed to spinal cord and muscle whereas XFD-expressing cells were excluded from the CNS and found in tissues such as the epidermis and fin. These results suggest that Fgf signalling is required for vegetal ectoderm to contribute to caudal neural tissue. They also suggest that the consequences of suppression of Fgf signalling in cells in dorsal and ventral domains of the vegetal ectoderm are different: dorsally, cells with compromised Fgf signalling frequently move into anterior neural tissue; ventrally, cells move into the prospective epidermis and are excluded from neural tissue. These results are consistent with analyses of embryos in which XFD is expressed ubiquitously and which show loss of posterior neural structures and anteriorisation of remaining CNS tissue on the dorsal side of the embryo (Kudoh, 2004).

Gastrulation generates mesoderm and endoderm from embryonic epiblast; soon after, the neural plate is established within the epiblast-both events require FGF signaling. A zinc finger transcriptional activator, Churchill (ChCh), is described that acts as a switch between different roles of FGF. FGF induces ChCh slowly; this activates Smad-interacting-protein-1 (Sip1), which blocks further induction of the mesoderm markers brachyury and Tbx6L by FGF. ChCh is first expressed as cells stop migrating through the primitive streak, and it regulates cell ingression. A simple mechanism is proposed by which FGF sensitizes cells to BMP signals. These results reveal that neural induction requires cessation of mesoderm formation at the midline in addition to the decision between epidermis and neural plate (Sheng, 2003).

FGF signaling in early epithelial differentiation in embryonic stem cell derived embryoid bodies

The role of FGF signaling in early epithelial differentiation was investigated in ES (embryonic stem) cell derived embryoid bodies. A dominant negative fibroblast growth factor receptor (FGFR) mutation was created by stably introducing into ES cells an Fgfr2 cDNA, truncated in its enzymatic domains. These cells failed to differentiate into cystic embryoid bodies. No epithelial differentiation and cavitation morphogenesis could be observed in the mutant, although its rate of cell proliferation remained unchanged. This phenotype was associated with a significant decrease in the activation of Akt/PKB and PLCgamma-1, as compared to the wild type, while the activation of MAPK/Erk was less affected. Requirement for PI 3-kinase signaling in embryoid body differentiation was demonstrated by specific inhibitors. Akt/PKB activation was abrogated by wortmannin in short-term experiments. In long-term cultures Ly294002 inhibited the differentiation of ES cells into embryoid bodies. These data demonstrate that for early epithelial differentiation FGF signaling is required through the PI 3-kinase-Akt/ PKB pathway (Chen, 2000).

Fibroblast growth factors and receptors are intimately connected to the extracellular matrix by their affinity to heparan sulfate proteoglycans. They mediate multiple processes during embryonic development and adult life. In this study, embryonic stem cell-derived embryoid bodies were used to model fibroblast growth factor signaling during early epithelial morphogenesis. To avoid redundancy caused by multiple receptors, a dominant negative mutation of Fgfr2 was employed. Mutant-derived embryoid bodies failed to form endoderm, ectoderm, and basement membrane and did not cavitate. However, in mixed cultures they displayed complete differentiation induced by extracellular products of the normal cell. At least one of these products is the basement membrane or factors connected to it. In the mutant, collagen IV and laminin-1 synthesis is coordinately suppressed. The basement membrane is required for embryoid body differentiation by rescuing columnar ectoderm differentiation and cavitation in the mutant by externally added basement membrane proteins. This treatment induced transcription of Eomesodermin, an early developmental gene, suggesting that purified basement membrane proteins can activate inherent developmental programs. These results provide a new paradigm for the role of fibroblast growth factor signaling in basement membrane formation and epithelial differentiation (Li, 2001).

FGF and trophoblast stem cell proliferation

The trophoblast cell lineage is essential for the survival of the mammalian embryo in utero. This lineage is specified before implantation into the uterus and is restricted to form the fetal portion of the placenta. A culture of mouse blastocysts or early postimplantation trophoblasts in the presence of fibroblast growth factor 4 (FGF4) permits the isolation of permanent trophoblast stem cell lines. These cell lines differentiated to other trophoblast subtypes in vitro in the absence of FGF4 and exclusively contribute to the trophoblast lineage in vivo in chimeras. It is concluded that trophoblast stem cell proliferation is promoted by FGF4 (Tanaka, 1998).

Little is known about how growth factors control tissue stem cell survival and proliferation. Mice with a null mutation of Shp2 (Ptpn11), a key component of receptor tyrosine kinase signaling, were analyzed. Null embryos die peri-implantation, much earlier than mice that express an Shp2 truncation. Shp2 null blastocysts initially develop normally, but they subsequently exhibit inner cell mass death, diminished numbers of trophoblast giant cells, and failure to yield trophoblast stem (TS) cell lines. Molecular markers reveal that the trophoblast lineage, which requires fibroblast growth factor-4 (FGF4), is specified but fails to expand normally. Moreover, deletion of Shp2 in TS cells causes rapid apoptosis. Shp2 is required for FGF4-evoked activation of the Src/Ras/Erk pathway that culminates in phosphorylation and destabilization of the proapoptotic protein Bim. Bim depletion substantially blocks apoptosis and significantly restores Shp2 null TS cell proliferation, thereby establishing a key mechanism by which FGF4 controls stem cell survival (Yang, 2006).

Distinct GATA6- and laminin-dependent mechanisms function downstream of FGF to regulate endodermal and ectodermal embryonic stem cell fates

The establishment of alternative cell fates during embryoid body differentiation has been investigated, when embryonic stem (ES) cells diverge into two epithelia simulating the pre-gastrulation endoderm and ectoderm. Endoderm differentiation and endoderm-specific gene expression, such as expression of laminin 1 subunits, is controlled by GATA6 induced by FGF. Subsequently, differentiation of the non-polar primitive ectoderm into columnar epithelium of the epiblast is induced by laminin 1. Using GATA6 transformed Lamc1-null endoderm-like cells, it was demonstrated that laminin 1 exhibited by the basement membrane induces epiblast differentiation and cavitation by cell-to-matrix/matrix-to-cell interactions that are similar to the in vivo crosstalk in the early embryo. Pharmacological and dominant-negative inhibitors reveal that the cell shape change of epiblast differentiation requires ROCK, the Rho kinase. Pluripotent ES cells display laminin receptors; hence, these stem cells may serve as target for columnar ectoderm differentiation. Laminin is not bound by endoderm derivatives; therefore, the sub-endodermal basement membrane is anchored selectively to the ectoderm, conveying polarity to its assembly and to the differentiation induced by it. Unique to these interactions is stem cell flow through two cell layers connected by laminin 1 and stem cell involvement in the differentiation of two epithelia from the same stem cell pool: one into endoderm controlled by FGF and GATA6; and the other into epiblast regulated by laminin 1 and Rho kinase (Li, 2004).

The inner cell mass (ICM) of preimplantation and early postimplantation mammalian embryos contain cells ancestral to the entire individual, that undergo extensive morphological change prior to gastrulation. In the blastocyst and early egg cylinder the ICM consists of an aggregate of non-polar stem cells, which before gastrulation undergo epithelialization and cavitation, creating a pseudostratified columnar epithelium that surrounds a central cavity similar to the proamniotic canal of the early embryo. The pseudostratified columnar epithelium or epiblast attaches to the sub-endodermal basement membrane (BM). This polarized epithelium allows intermingling of clonal derivatives and is thought to be necessary for gastrulation. Much is known about the role of endoderm to ectoderm signalling in anteroposterior patterning of the early embryo. The establishment of major elements of the amniote body plan during gastrulation has been also studied in detail. However, the mechanism that precedes these changes and transforms the non-polar primitive ectoderm into the columnar polar epiblast is little understood (Li, 2004).

Embryonic stem cell derived embryoid bodies (EBs) are similar to the egg cylinder embryo, but, in contrast to it, they can be grown in large quantities, providing a useful model for early embryogenesis. The mechanism of EB differentiation has been set out as a model for pregastrulation development and tube formation by cavitation. EBs have an external endoderm that is similar to the primitive or visceral endoderm of the embryo and is separated from the inner columnar ectoderm by a basement membrane (BM). Using a genetically undefined spontaneous mutation, which fails to form the columnar ectoderm layer, it was proposed that cavitation is regulated by two signals: one emanating from the outer endoderm layer was thought to be responsible for the apoptotic signal/s of cavitation; the second, originating in the BM, was considered necessary for the maintenance and survival of the columnar ectoderm (Li, 2004 and references therein).

The work carried out in this study started as a study of the role of FGF signalling in EB differentiation and led to questions regarding BM assembly that were investigated using ES cells that express truncated Fgfr2 cDNA as a dominant-negative mutation. ES cells expressing dnFgfr fail to develop the two characteristic cell layers of the EB. They display a homogenous aggregate of non-polar cells and form no endoderm or ectoderm-like elements, but survive for weeks during cultivation. EBs formed by dnFgfr ES cells fail to synthesize laminin and collagen IV isotypes, which supply the protein network of the BM. Co-cultivating wild-type and dnFgfr ES cells rescued EB differentiation, suggesting that an FGF-controlled extracellular substance, subsequently identified as laminin 1, is required for epiblast differentiation. Exogenously added laminin 1 partially rescues the EB phenotype and induces epithelial transformation, demonstrating that laminin 1 produced by the endoderm is necessary and sufficient to induce epiblast polarization (Li, 2004).

Laminin 1 has been shown to be required for EB differentiation. Targeted disruption of ß1-integrin, which inhibits laminin alpha1 synthesis, interferes with epiblast differentiation. Disruption of Lamc1 encoding laminin gamma1, one of the three polypeptides of the laminin 1 heterotrimer, leads to a similar phenotype. Significantly, defective epiblast differentiation caused by loss of either gene was rescued by exogenously added laminin 1, which in turn could be inhibited by the E3 fragment of laminin alpha1 containing the heparin and sulfatide binding site of the LG4 globular domain of the laminin alpha1-chain. Recognising the potential importance of these findings for understanding epithelial differentiation and early development, it would help their analysis if the succession and main intermediates of EB differentiation were defined (Li, 2004).

In the present study, attempts were made to obtain a comprehensive view of the developmental interactions that precede gastrulation. To achieve this, several specific questions had to be answered. Is FGF signalling required for the differentiation of both epithelia and the pattern of their arrangement in the EB, or for only an initial step that is necessary for later events? Defective FGF signalling could be partially restored by exogenous laminin 1. The next question is can the same effect be obtained by laminin 1 presented by the BM in a physiological cell-matrix interaction? It was also important to determine whether laminin affects the stem cell directly, or whether it activates precursors after they reached a specific stage of FGF dependent differentiation. To answer these questions, mutant and wild-type ES cell lines were used , and their behaviour was studied as an effect of chemical inhibitors and co-cultivation experiments between mutant and wild-type cells (Li, 2004).

As an experimental system to elucidate interactions between the endoderm and primitive ectoderm GATA4- or GATA6-transformed endoderm-like cells co-cultivated with mutant ES cell lines were used. This system demonstrated that GATA4 and GATA6 transform ES cells into functional extra-embryonic endoderm that deposits a BM, which in turn mediates epiblast polarization. GATA transformed cells synthesize and later secrete laminin 1 and collagen IV into the culture supernatant, which could be used to rescue epiblast differentiation. Genetic evidence of laminin gamma1 null ES cells has demonstrated the specificity of mutant rescue. This experimental system thus recreated the physiological BM-mediated interaction and allowed the separation of endoderm and epiblast differentiation according to their respective FGF/GATA6 and laminin/Rho kinase-dependent mechanisms (Li, 2004).

Endoderm differentiation depends on FGF signalling, as demonstrated by the targeted disruption of Fgf4. Fgf4 is expressed in the ICM and contributes to the maintenance of the endoderm, where the multiple FGF receptors that read its signals are localized. Expression of GATA4 and GATA6, where GATA4 is regulated by GATA6, is controlled by FGF signalling. Nevertheless, the immediate downstream elements of FGF signalling are insufficiently understood in EB differentiation. In vitro evidence suggests that most FGF dependent signals go through Frs2a, a docking protein, which communicates with the Grb2 adaptor. Interestingly although null mutants of Fgf4 die with defective endoderm development shortly after implantation, Frs2a null embryos survive until advanced gastrulation, indicating that FGF signalling may exhibit unique characteristics in the early embryo. Analysis of signal transduction in dnFgfr ES cells revealed that PI3K-Akt/PKB rather than MAPK-ERK signalling is affected by defective FGF activity. In agreement, this study found that constitutively active Akt/PKB enhances endoderm development and the synthesis of laminin and collagen IV isotypes, indicating that the PI3K-Akt/PKB pathway predominates in FGF-dependent endoderm differentiation (Li, 2004).

GATA6 is an intermediary of FGF signalling. GATA6, which is transcribed already in the ICM, behaves as a master gene for endoderm differentiation. GATA6 activates the synthesis of all three polypeptide chains of laminin 1, which together with collagen IV, nidogen and perlecan assemble into the sub-endodermal BM. GATA factors induce endoderm differentiation and BM assembly even in dnFgfr ES cells, indicating that once activated, these transcription factors induce endoderm differentiation independently from FGF signalling. Because endoderm differentiation requires GATA6 and because cysts of GATA6 transformed cells contain only endoderm-like elements, it is concluded that GATA factors are required and sufficient to induce endoderm development and deposition of the subendodermal BM (Li, 2004).

Additional elements of this pathway are the transcription factors COUP-TFs I and II, which are upregulated by GATA4/6 during endoderm development and induce Lamc1 and Lamb1 expression. It follows that minimal elements of this interaction are, sequentially, Fgf4, multiple Fgfr, PI3K and AKT/PKB, GATA6 and GATA4, COUP-TFs I and II, as well as the genes encoding the three polypeptide chains of laminin 1 (Li, 2004).

Evidence demonstrates that E-cadherin is also required for early EB differentiation. E-cadherin-null ES cells fail to aggregate, do not form a normal ectoderm and do not undergo EB differentiation. Therefore, E-cadherin-dependent ES cell aggregation may be a prerequisite for the restriction of FGF signalling to the outer cells of the developing EB. E-cadherin is connected to the ß-catenin-GSK3-wnt pathway. Patterning events involving cadherin-Wnt/ß-catenin interactions have been shown to be controlled by FGF signalling (Li, 2004).

There is strong evidence for the epithelialization of ES cells by exogenous laminin 1. Laminin 1 can induce epiblast differentiation as part of the BM that mediates the physiological interaction of the endoderm with the epiblast. While laminin 1 binds to ES cells and their ectodermal derivatives, it does not associate with the primitive endoderm. Thus, the cell-binding domains of the laminin alpha1 chain determine the location of the subendodermal BM by interacting with their receptors displayed by the stem cells localized below the endoderm layer. This therefore defines the direction of laminin-mediated signalling, thereby determining the topographical relationship of endoderm and ectoderm (Li, 2004).

Besides inducing epiblast polarization, the BM affects the simple two-cell layer pattern of the EB and egg cylinder embryo. Since cell-to-matrix interactions take place through direct contact, epithelialization of residual stem cells is precluded, and a single epiblast monolayer develops from cells immediately adjacent to the BM. It has been proposed that the residual stem cells are removed by programmed cell death induced by factors derived from the endoderm, to form a central cavity. Investigation of the role of BMP signalling in cavitation indicates that BMP2 synthesizes in the endoderm, and BMP4 in the primitive ectoderm can both contribute to cavitation, although BMP4 is expressed only for a short period. The data indicate that cavitation and columnar ectoderm differentiation do not require the endoderm, provided that exogenous laminin 1 is presented. It is therefore possible that the developing ectoderm itself secretes the necessary apoptotic factors, such as BMP4, although inhibition of ROCK activity uncouples cavitation from full epithelialization of the primitive ectoderm and argues that cavitation may be either not different from necrosis, or it might be due to mechanical separation of the columnar ectoderm from the residual stem cells. This issue requires further study (Li, 2004).

Dominant-negative ROCK abolishes epiblast polarization without affecting endoderm differentiation, suggesting that it may be regulated separately in the two cell lineages. This assumption was supported by observing that ROCK expression and epiblast polarization does not require the endoderm for the laminin-induced differentiation of dnFgfr ES cells. Although ROCK is required for the epithelialization of the primitive ectoderm, it is not sufficient to induce this process, as suggested by the observation that dominant-active ROCK does not rescue dnFgfr differentiation. Although in the epiblast ROCK activity may be induced by laminin, in the endoderm it appears to be under FGF control and the resistance of endodermal differentiation to ROCK inhibition is consistent with the possibility that RAC1 or Cdc42, which are co-expressed in the endoderm, may have a role in endodermal differentiation (Li, 2004).

Separation of endoderm and epiblast differentiation has been repeatedly observed in this study. FGF signalling is shown to be required for endoderm differentiation but not for epiblast polarization, which is independently induced by laminin 1 of the sub-endodermal BM. The two lineages are also distinguished by laminin binding. ES cells and their ectodermal derivatives bind laminin, while the primitive and visceral endoderm do not, which defines the direction of laminin-induced differentiation. It follows that the extra-embryonic and embryonic epithelium of the EB and egg cylinder embryo develop by distinct mechanisms, which are connected by the inductive activity of the laminin component of their common BM. Future research will have to clarify whether other epithelial transitions are also controlled by laminin-dependent mechanisms (Li, 2004).

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