Interactive Fly, Drosophila

Role of FGF during gastrulation (part 1/2)

Mesoderm induction is a critical early step in vertebrate development, involving changes in gene expression and morphogenesis. In Xenopus, normal mesoderm formation depends on signalling through the fibroblast growth factor (FGF) tyrosine kinase receptor. One important signalling pathway from receptor tyrosine kinases involves p21ras. Ras associates with the serine kinase c-Raf-1 in a GTP-dependent manner, and this complex phosphorylates and activates MAPK/ERK kinase (MEK), a protein kinase with dual specificity. MEK then activates p42mapk and (at least in mammals) p44mapk, both members of the mitogen-activated protein (MAP) kinase family. FGF activates MAP kinase during mesoderm induction: the use of dominant-negative constructs suggests that mesoderm induction by FGF requires both Ras and Raf. However, these experiments do not reveal whether Ras and Raf do act through MAP kinase to induce mesoderm or whether another pathway, such as the phosphatidylinositol 3-kinase cascade, is involved. It is shown that expression of active forms of MEK or of MAP kinase induces ventral mesoderm of the kind elicited by FGF. Overexpression of a Xenopus MAP kinase phosphatase blocks mesoderm induction by FGF, and causes characteristic defects in mesoderm formation in intact embryos, whereas inhibition of the P13 kinase and p70 S6 kinase pathways has no effect on mesoderm induction by FGF. FGF induces different types of mesoderm in a dose-dependent manner; strikingly, this is mimicked by expressing different levels of activated MEK. Together, these experiments demonstrate that activation of MAP kinases is necessary and sufficient for mesoderm formation (Umbhauer, 1995).

Fibroblast growth factor (FGF) signaling is required prior to gastrulation in the mouse embryo. To test for the spatial and temporal requirements of FGF signaling, a dominant negative FGF receptor (dnFGFR) was used to make transgenic mouse embryos. In mosaic embryos, cell division ceases at the fifth cell division in all cells that express the mutant receptor, but cell death does not increase. After the fifth cell division, the progeny of unaltered cells and cells expressing lacZ continue to accumulate at the same rate, suggesting that the FGF requirement is cell autonomous. In mosaic embryos, lacZ, but not dnFGFR expression is detected in mitotic trophoblasts adjacent to the ICM. Conversely, dnFGFR-expressing extraembryonic ectoderm cells are detected at the abembryonic pole in postmitotic cells. In blastocysts expressing the dnFGFR in all cells, the morphology appears normal and inner cell masses (ICMs) form, but the resultant embryos have only one-third the number of cells as compared to control embryos. In these blastocysts, cell division has also ceased at the fifth cell division, but cavitation, a concurrent morphogenetic event, initiates and progresses normally. To test for the continuing requirement of FGF, FGFR-3 was overexpressed in all cells. This resulted in an increase in cell numbers after the fifth cell cycle. In an experimental model system for postimplantation development, a continuing role for FGF is suggested: the addition of FGF-4 to blastocyst outgrowths increases the number of extraembryonic ectoderm cells. Thus, FGF signaling induces the cell division of embryonic and extraembryonic cells in the preimplantation mouse embryo starting at the fifth cell division. The signal requirement for FGF is cell autonomous, but is not required to prevent cell death. This provides the first evidence for the necessity of a growth factor before implantation (Chai, 1998).

The Brachyury (T) gene (see Drosophila Brachyenteron) is required for the formation of posterior mesoderm and for axial development in both mouse and zebrafish embryos. In these species, and in Xenopus, the gene is expressed transiently throughout the presumptive mesoderm; transcripts then persist in notochord and posterior tissues. In Xenopus embryos, expression of the Xenopus homolog of Brachyury (Xbra) can be induced in presumptive ectoderm by basic fibroblast growth factor (FGF) and activin; in the absence of functional FGF or activin signaling pathways, expression of the gene is severely reduced. Ectopic expression of Xbra in presumptive ectoderm causes mesoderm to be formed. As Brachyury and its homologs encode sequence-specific DNA-binding proteins, it is likely that each functions by directly activating downstream mesoderm-specific genes. Expression in Xenopus embryos of RNA encoding a dominant-negative FGF receptor inhibits the mesoderm-inducing activity of Xbra. Ectopic expression of Xbra is shown to activate transcription of the embryonic FGF gene, and embryonic FGF can induce expression of Xbra. This suggests that the two genes are components of a regulatory loop. Consistent with this idea, dissociation of Xbra-expressing cells causes a dramatic and rapid reduction in levels of Xbra, but the reduction can be inhibited by the addition of FGF. It is concluded that formation of mesoderm tissue requires an intact FGF signaling pathway downstream of Brachyury. This requirement is due to a regulatory loop, in which Brachyury activates the expression of a member of the FGF family, and FGF maintains expression of Brachyury (Schulte-Merker, 1995).

In addition to a role in mesoderm induction during blastula stages, FGF signalling plays an important role in maintaining the properties of the mesoderm in the gastrula of Xenopus laevis. eFGF is a maternally expressed secreted Xenopus FGF with potent mesoderm-inducing activity. However, it is most highly expressed in the mesoderm during gastrulation, suggesting a role after the period of mesoderm induction. eFGF is inhibited by the dominant negative FGF receptor. Embryos overexpressing the dominant negative receptor show a change of behaviour of the dorsal mesoderm such that it moves around the blastopore lip instead of elongating in an antero-posterior direction. In such embryos there is a reduction in Xbra expression during gastrulation. During blastula stages eFGF and Xbra are able to activate one another's expression, suggesting that they are components of an autocatalytic regulatory loop. Xbra expression in isolated gastrula mesoderm cells is maintained by eFGF, suggesting that eFGF continues to regulate the expression of Xbra in the blastopore region. In addition, overexpression of eFGF after the mid-blastula transition results in the up-regulation of Xbra expression during gastrula stages and causes suppression of the head and enlargement of the proctodeum, which is the converse of the posterior reductions of the FGF dominant negative receptor phenotype. These data suggest an important role for eFGF in regulating the expression of Xbra and for the eFGF-Xbra regulatory pathway in the control of mesodermal cell behaviour during gastrula stages (Isaacs, 1994).

The transcriptional activity of a set of genes, which are all expressed in overlapping spatial and temporal patterns within the Spemann organizer of Xenopus embryos, can be modulated by peptide growth factors. Xegr-1, a zinc finger protein-encoding gene, has been identified as a novel member of this group of genes. The spatial expression characteristics of Xegr-1 during gastrulation are most similar to those of Xbra. Making use of animal cap explants, analysis of the regulatory events that govern induction of Xegr-1 gene activity reveals that, in sharp contrast to transcriptional regulation of Xbra, activation of Ets-serum response factor (SRF) transcription factor complexes is required and sufficient for Xegr-1 gene expression. The Ets-SRF complexes are known to act downstream of the MAP kinase pathway, and in the case of Xegr-1 the complex is shown to function downstream of FGF signaling. The finding that Xegr-1 activation requires Ets-SRF complexes provides the first indication for Ets-SRF complexes binds to serum response elements that are activated during gastrulation. MAP kinase signaling cascades can induce and sustain expression of both Xegr-1 and Xbra. Ectopic Xbra is found to induce Xegr-1 transcription by an indirect mechanism that appears to operate via primary activation of fibroblast growth factor secretion. These findings define a cascade of events that links Xbra activity to the activation of FGF signaling and the subsequent signal-regulated control of Xegr-1 transcription in the context of early mesoderm induction in Xenopus laevis (Panitz, 1998).

The ability of Brachyury to activate transcription is essential for its biological function, but nothing is known about its target genes. Xenopus Brachyury is shown to directly regulate expression of eFGF by binding to an element positioned ~1 kb upstream of the eFGF transcription start site. Activation of Xbra by eFGF occurs through the MAP kinase pathway and requires neither cell-cell communication nor protein synthesis. The hormone-inducible Xbra construct Xbra-GR was used to determine whether, according to the same criteria, activation of eFGF by Xbra is direct. Xbra-GR does induce expression of eFGF in dispersed cells, and in the presence both of cycloheximide and of a dominant-negative FGF receptor. Thus, induction of eFGF by Xbra is cell autonomous, does not involve synthesis of an intermediate transcription factor and does not require an intact FGF signaling pathway. This is in contrast with autoinduction of Xbra, which is inhibited by cell dispersion and by a dominant-negative FGF receptor and presumably occurs via eFGF (Casey, 1998).

The data above indicate that Xbra activates expression of eFGF directly. 2.5 kb of the upstream regulatory region of eFGF were isolated. Sequencing revealed a single 10 base pair element TTTCACACCT located 936 nucleotides upstream of the transcription start site. This sequence is identical to half of the 20 base pair palindromic Brachyury site previously identified. A related sequence, AACCACACCT, is located 123 nucleotides downstream of the transcription start site. Previous reports have suggested that Brachyury does not bind to a half-palindrome, and that two half-palindromes, appropriately spaced, are required for transcription activation. However, the 5' regulatory regions of mouse and human FGF-4, to which eFGF is closely related, also contain a single Brachyury half-site within about 1 kb of their transcription start sites. This conservation suggests that the sequence is involved in regulation of eFGF/FGF-4 expression. Xbra is shown to bind specifically to the Brachyury half-site as well as to the complete palindrome. Both half-sites are required for full induction of the 2.5 kb eFGFpromoter by Xbra (Casey, 1998).

These experiments are consistent with the idea that Xbra and eFGF are components of an indirect autoregulatory loop in which each maintains expression of the other. The data also indicate that this loop functions predominantly in notochord and dorsal mesoderm, because inhibition of Xbra function results in loss of expression of eFGF and Xbra itself in these tissues. The same may be true in the mouse embryo, where there is no evidence for direct interaction between Brachyury and FGF family members in the primitive streak, but it remains possible that an autoregulatory loop functions in the notochord and head process (Casey, 1998 and references).

Fgf8 and Fgf4 encode FGF family members that are coexpressed in the primitive streak of the gastrulating mouse embryo. The phenotype of Fgf8-/- embryos were analyzed and it was discovered that they fail to express Fgf4 in the streak. In the absence of both FGF8 and FGF4, epiblast cells move into the streak and undergo an epithelial-to-mesenchymal transition, but most cells then fail to move away from the streak. As a consequence, no embryonic mesoderm- or endoderm-derived tissues develop, although extraembryonic tissues form. Patterning of the prospective neuroectoderm is greatly perturbed in the mutant embryos. Anterior neuroectoderm markers are widely expressed, at least in part because the anterior visceral endoderm, which provides signals that regulate marker expression, is not displaced proximally in the absence of definitive endoderm. Posterior neuroectoderm markers are not expressed, presumably because there is neither mesendoderm underlying the prospective neuroectoderm nor a morphologically normal node to provide the inductive signals necessary for their expression. This study identifies Fgf8 as a gene essential for gastrulation and shows that signaling via FGF8 and/or FGF4 is required for cell migration away from the primitive streak (Sun, 1999).

In invertebrates, FGF signaling is also necessary for cell migration. For example, in Drosophila it is required for migration and spreading of the embryonic mesoderm over the ectoderm and for branching morphogenesis of the tracheal system, and in Caenorhabditis elegans it is required for sex myoblast migration. In both organisms, ectopic expression experiments have suggested that FGFs can function as attractants for cell migration. By analogy, one might argue that FGF8 produced in the VE acts as an attractant for cell migration away from the streak. This hypothesis was tested by injecting wild-type embryonic stem cells into Fgf8 minus blastocysts, producing chimeras in which the VE presumably contained only Fgf8 minus cells, whereas the epiblast contained a mixture of wild-type and mutant cells. No defects in gastrulation were detected in four such chimeras, in which at least 25% of the embryonic cell population was derived from wild-type ES cells. In these embryos, Fgf8 minus cells contributed to all tissues, including somites, head mesenchyme, and foregut. These results indicate that lack of FGF8 in the VE is not responsible for the gastrulation defects in Fgf8 mutant embryos. Instead, FGF signaling appears to be required in the primitive streak itself, presumably to regulate the production of proteins necessary for cell migration (Sun, 1999).

Genes that encode molecules involved in adhesive interactions between cells and their surrounding extracellular matrix (ECM) are obvious candidates for the downstream targets affected by loss of Fgf8 function. Mutational analysis has shown that there is a deficit of mesoderm in embryos homozygous for null alleles of Fibronectin, Integrin alpha5, and Focal adhesion kinase, which encode a component of the ECM, part of the receptor for Fibronectin, and a nonreceptor tyrosine kinase thought to mediate Integrin signaling, respectively. This suggests that those genes might be required for cell migration away from the primitive streak. However, abnormalities in the mutant embryos are not detected until at least the late headfold stage. It therefore seems unlikely that the more severe phenotype of Fgf8 minus embryos is due to effects of FGF8/4 signaling on any one of these genes, although it remains possible that the defects are due to simultaneous effects on more than one such gene (Sun, 1999 and references).

Another type of molecule that appears to play some role in cell migration away from the primitive streak is the transcription factor T. When the behavior of T null homozygous cells is monitored in chimeras, they are found to accumulate in the mesodermal layer of the streak region, but this effect is not evident until the headfold stage. Moreover, T null mutant embryos do not show any obvious defects at the primitive streak stages. The fact that Fgf8 mutant embryos display a more severe phenotype argues against interference with T expression as the primary cause of the defect in cell migration. However, other genes related to T might be the downstream targets of FGF signaling required for cell migration away from the streak. Consistent with this hypothesis, it was found that FGF8/4 signaling regulates expression of at least some T-related genes. For example, Tbx6 is not expressed in Fgf8 minus embryos. Furthermore, although T expression is detected in epithelial cells in the mutant streak region, it is not detected in nascent mesenchymal cells that have traversed the streak and accumulated there. In contrast, T expression is detected in both the epithelial and mesenchymal portions of the streak in Fgf8+ embryos, and even in cells a short distance away from the streak. These observations are consistent with some aspects of the positive-feedback loop model proposed for regulation of the expression of Xbra and eFgf, the Xenopus orthologs of T and Fgf4, respectively and the finding that in zebrafish, expression of T and two T-related genes, spadetail and Tbx6 is regulated by FGF signaling (Sun, 1999 and references).

Signals released from Spemann's organizer, together with ventralizing factors such as BMPs, are necessary to pattern the dorsoventral axis of the vertebrate embryo. A member of the FGF family, FGF-8, is not secreted by the axial mesoderm but is expressed in a dorsoventral gradient at the margin of the zebrafish gastrula, and contributes to the establishment of the dorsoventral axis of the embryo. Ectopic expression of FGF-8 leads to the expansion of dorsolateral derivatives at the expense of ventral and posterior domains. FGF-8 displays some organizer properties as it induces the formation of a partial secondary axis in the absence of factors released from Spemann's organizer territory. Analysis of its interaction with the ventralizing factors, BMPs, reveals that overexpression of FGF-8 inhibits the expression of these factors in the ventral part of the embryo as early as blastula stage, suggesting that FGF-8 acts upstream of BMP2 and BMP4. It is concluded that FGF-8 is involved in defining dorsoventral identity and is an important organizing factor responsible for specification of mesodermal and ectodermal dorsolateral territories of the zebrafish gastrula (Furthauer, 1997).

Interactions between Nodal/Activin and Fibroblast growth factor (Fgf) signalling pathways have long been thought to play an important role in mesoderm formation. However, the molecular and cellular processes underlying these interactions have remained elusive. This study addresses the epistatic relationships between Nodal and Fgf pathways during early embryogenesis in zebrafish. (1) Fgf signalling is found to be required downstream of Nodal signals for inducing the Nodal co-factor One-eyed-pinhead (Oep). Thus, Fgf is likely to be involved in the amplification and propagation of Nodal signalling during early embryonic stages. This could account for the previously described ability of Fgf to render cells competent to respond to Nodal/Activin signals. In addition, overexpression data shows that Fgf8 and Fgf3 can take part in this process. (2) Combining zygotic mutations in ace/fgf8 and oep disrupts mesoderm formation, a phenotype that is not produced by either mutation alone and is consistent with this model of an interdependence of Fgf8 and Nodal pathways through the genetic regulation of the Nodal co-factor Oep and the cell propagation of Nodal signalling. Moreover, mesodermal cell populations are affected differentially by double loss-of-function of Zoep;ace. Most of the dorsal mesoderm undergoes massive cell death by the end of gastrulation, in contrast to either single-mutant phenotype. However, some mesoderm cells are still able to undergo myogenic differentiation in the anterior trunk of Zoep;ace embryos, revealing a morphological transition at the level of somites 6-8. Further decreasing Oep levels by removing maternal oep products aggravates the mesodermal defects in double mutants by disrupting the fate of the entire mesoderm. Together, these results demonstrate synergy between oep and fgf8 that operates with regional differences and is involved in the induction, maintenance, movement and survival of mesodermal cell populations (Mathieu, 2004).

The establishment of dorsoventral (DV) patterning in vertebrate embryos depends on the morphogenic activity of a group of Tgfß superfamily members, the bone morphogenetic proteins (Bmps) (which specify ventral cell fates), and on their interaction with their dorsally secreted cognate inhibitors chordin and noggin. In the zebrafish, genetic analysis has revealed that Bmp2b and Bmp7, as well as their antagonist chordin, are required for proper DV patterning. The expression of Bmp genes is initially activated in the whole blastula. Well before the beginning of gastrulation, Bmp gene expression progressively disappears from the dorsal side to become restricted to the ventral part of the embryo. This early restriction of Bmp gene expression, which occurs independently of noggin and chordin, is an essential step in the establishment of DV patterning. The progressive ventral restriction of Bmp gene transcripts is coincident with the spreading of Fgf activity from the dorsal side of the embryo, suggesting that Fgf signalling is implicated in dorsal downregulation of Bmp gene expression. In accordance with this, activation of the Fgf/Ras/Mapk-signalling pathway inhibits ventral Bmp gene expression, thereby causing a dorsalisation of the embryo. Conversely, inhibition of Fgf signalling causes Bmp gene expression to expand dorsally, leading to an expansion of ventral cell fates. In accordance with an important role of Fgf signalling in the DV patterning of the zebrafish, loss of Fgf8 function enhances the ventralisation of chordin-deficient embryos. These results thereby demonstrate that pre-gastrula stage Fgf-signalling is essential to delimit the expression domain of the genes encoding the functional morphogen of the dorsoventral axis of the early zebrafish embryo (Fürthauer, 2004).

Table of contents

branchless: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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