Interactive Fly, Drosophila

Extracellular matrix and FGF

Two FGFR binding sites on bFGF act in concert to initiate signal transduction. Both FGFR binding surfaces are distinct from the heparin sulfate proteoglycan binding domain. The higher affinity primary binding interaction comprises a cluster of solvent exposed hydrophobic amino acids (Tyr-24, Tyr-103, Leu-140, and Met-142), and two polar residues (Arg-44 and Asn-101). The hydrophobic contacts dominate the primary binding interaction and provide approximately 75% of the binding affinity. The secondary FGFR binding site on bFGF has an approximately 250-fold lower affinity and is composed of amino acids Lys-110, Tyr-111, and Trp-114 in a surface-exposed type I beta-turn (formerly known as the putative receptor binding loop). Binding of FGFR to both bFGF surfaces in a stoichiometry of 2FGFR:1bFGF is required for growth factor mediated cell proliferation. This represents a mechanism for the fibroblast growth factor receptor family in which FGF, acting as a monomeric ligand, facilitates FGFR dimerization and subsequent signal transduction events (Springer, 1994).

Heparan sulfate proteoglycans (HSPG) have been shown to be involved in the activation of tyrosine kinase receptors by basic fibroblast growth factor (bFGF), a strong inhibitor of skeletal muscle differentiation. Skeletal muscle fibers contact extracellular matrix (ECM) that surrounds individual fibers (endomysium) and bundles of several fibers (perimysium). Perlecan is an HSPG present in the majority of basement membranes. The amount of HSPG that can be immunoprecipitated decreases with muscle differentiation. Perlecan is localized on the myoblast surface and is associated with patches of incipient extracellular matrix. The expression of perlecan mRNA decreases substantially during skeletal muscle differentiation, in contrast to the increase in transcripts for specific skeletal muscle proteins, such as myogenin and creatine kinase. Almost no perlecan staining associated with the surface of myotubes is observed. All these results suggests that perlecan, an HSPG that binds myogenic inhibitory bFGF, normally associated with basement membranes in adult tissues, is present on the surface of myoblasts and its expression is down regulated during skeletal muscle differentiation (Larrain, 1997).

Heparan sulfate (HS) is abundant in the developing brain and is a required co-factor for many types of fibroblast growth factor (FGF) signaling in vitro. Some HSs, when added exogenously to the developing Xenopus optic pathway, severely disrupt target recognition, causing axons from the retina to bypass their primary target, the optic tectum. Significantly, HS sidechains from a neuroepithelial perlecan variant that preferentially bind FGF-2, HS(FGF-2), cause aberrant targeting, whereas those that preferentially bind FGF-1 do not. Charge-matched fragments of HS(FGF-2) show that the mistargeting activity associates with the FGF-binding fragments. Heparitinase removal of native HSs at the beginning of optic tract formation retards retinal axon elongation; addition of FGF-2 restores axon extension but axons lose directionality. Late HS removal, after axons have extended through the tract, elicits a tectal bypass phenotype indicating a growth promoting and guidance function for native HSs. These results demonstrate that different HS sidechains from the same core protein differentially affect axon growth in vivo, possibly due to their distinct FGF-binding preferences, and suggest that growth factors and HSs are important partners in regulating axon growth and guidance in the developing visual system (Walz, 1997).

Fibroblast growth factor (FGF)-1, also known as acidic FGF, is a multifunctional heparin-binding protein that is mitogenic for a wide variety of cell types cultured in vitro and a potent angiogenic agent in vivo. These cellular responses are mediated via high-affinity binding to a family of four membrane-spanning tyrosine kinase receptors. FGF-1-stimulated mitogenesis is potentiated by heparin, a sulfated glycosaminoglycan. The induction levels and temporal expression kinetics of two immediate-early response mRNAs (early growth response gene-1, thrombospondin-1) as well as two delayed-early response mRNAs (proliferin, ornithine decarboxylase) were monitored. Although FGF-1 alone can promote the initial induction of these four mRNAs, heparin coaddition is necessary for prolonged delayed-early mRNA expression. This heparin effect occurs when cells are stimulated with wild-type FGF-1 but not with FGF-1/glu132 (an FGF-1 mutant with a reduced apparent affinity for heparin). FGF-1 and heparin must be added together at the initial time of mitogen stimulation and they must remain present in the cell culture medium for a minimum period of 8 h to promote sustained delayed-early mRNA expression. These findings are consistent with the proposal that heparin promotes a long-term FGF-1:FGFR interaction which is required for sustained delayed-early gene expression and a full mitogenic response (Donohue, 1997).

Fibroblast growth factor (FGF) 1 and FGF-2 are prototypic members of the FGF family, which to date comprises at least 18 members. Surprisingly, even though FGF-1 and FGF-2 share more than 80% sequence similarity and an identical structural fold, these two growth factors are biologically very different. FGF-1 and FGF-2 differ in their ability to bind isoforms of the FGF receptor family as well as the heparin-like glycosaminoglycan (HLGAG) component of proteoglycans on the cell surface in order to initiate signaling in different cell types. There is now evidence for one mechanism by which these two proteins could differ biologically. Previously, it has been noted that FGF-1 and FGF-2 can oligomerize in the presence of HLGAGs. Therefore, whether FGF-1 and FGF-2 oligomerize by the same mechanism or by a different one was investigated. Under identical conditions, FGF-1 and FGF-2 differ in both degree and kind of oligomerization. Furthermore, an extensive analysis of FGF-1 and FGF-2 uncomplexed and HLGAG complexed crystal structures provides a ready explaination for why FGF-2 forms sequential oligomers whereas FGF-1 forms only dimers. FGF-2, which possesses an interface capable of protein association, forms a translationally related (side-by-side) oligomer, whereas FGF-1, which does not have this interface, forms only a symmetrically related dimer. This conserved association is such that the internal 3-fold axis of the molecule is approximately parallel, and the HLGAG chain can be easily accommodated to bridge neighboring FGF-2 molecules, thereby facilitating FGF-2 oligomerization. Taken together, these data show that FGF-1 and FGF-2, despite their sequence homology, differ in their mechanism of oligomerization (Venkataraman, 1999a).

Signaling by fibroblast growth factor 10 (FGF10) through FGFR2b is essential for lung development. Heparan sulfates (HS) are major modulators of growth factor binding and signaling present on cell surfaces and extracellular matrices of all tissues. Although recent studies provide evidence that HS are required for FGF-directed tracheal morphogenesis in Drosophila, little is known about the HS role in FGF10-mediated bud formation in the vertebrate lung. HS expression has been mapped in the early lung and how HS interactions with FGF10-FGFR2b influence lung morphogenesis has been examined. The data show that a specific set of HS low in O-sulfates is dynamically expressed in the lung mesenchyme at the sites of prospective budding near Fgf10-expressing areas. In turn, highly sulfated HS are present in basement membranes of branching epithelial tubules. Disrupting endogenous gradients of HS or altering HS sulfation in embryonic lung culture systems prevents FGF10 from inducing local responses and markedly alters lung pattern formation and gene expression. Experiments with selectively sulfated heparins indicate that O-sulfated groups in HS are critical for FGF10 signaling activation in the epithelium during lung bud formation, and that the effect of FGF10 in pattern is in part determined by regional distribution of O-sulfated HS. Moreover, expression of a HS 6-O-sulfotransferase preferentially at the tips of branching tubules is described. The data suggest that the ability of FGF10 to induce local budding is critically influenced by developmentally regulated regional patterns of HS sulfation (Izvolsky, 2003).

Heparan sulphate proteoglycans such as glypicans are essential modulators of intercellular communication during embryogenesis. In Xenopus laevis embryos, the temporal and spatial distribution of Glypican 4 (Gpc4) transcripts during gastrulation and neurulation suggests functions in early development of the central nervous system. The role of Xenopus Gpc4 has been functionally analyzed by using antisense morpholino oligonucleotides; Gpc4 is shown to be part of the signalling network that patterns the forebrain. Depletion of GPC4 protein results in a pleiotropic phenotype affecting both primary axis formation and early patterning of the anterior central nervous system. Molecular analysis shows that posterior axis elongation during gastrulation is affected in GPC4-depleted embryos, whereas head and neural induction are apparently normal. During neurulation, loss of GPC4 disrupts expression of dorsal forebrain genes, such as Emx2, whereas genes marking the ventral forebrain and posterior central nervous system continue to be expressed. This loss of GPC4 activity also causes apoptosis of forebrain progenitors during neural tube closure. Biochemical studies establish that GPC4 binds FGF2 and modulates FGF signal transduction. Inhibition of FGF signal transduction, by adding the chemical SU5402 to embryos from neural plate stages onward, phenocopies the loss of gene expression and apoptosis in the forebrain. It is proposed that GPC4 regulates dorsoventral forebrain patterning by positive modulation of FGF signalling (Gall, 2003).

Heparan sulphate proteoglycans (HSPGs) are known to be crucial for signalling by the secreted Wnt, Hedgehog, Bmp and Fgf proteins during invertebrate development. However, relatively little is known about their effect on developmental signalling in vertebrates. This study reports the analysis of daedalus, a novel zebrafish pectoral fin mutant. Positional cloning identified fgf10 as the gene disrupted in daedalus. fgf10 mutants strongly resemble zebrafish ext2 and extl3 mutants, which encode glycosyltransferases required for heparan sulphate biosynthesis. This suggests that HSPGs are crucial for Fgf10 signalling during limb development. Consistent with this proposal, a strong genetic interaction is observed between fgf10 and extl3 mutants. Furthermore, application of Fgf10 protein can rescue target gene activation in fgf10, but not in ext2 or extl3 mutants. By contrast, application of Fgf4 protein can activate target genes in both ext2 and extl3 mutants, indicating that ext2 and extl3 are differentially required for Fgf10, but not Fgf4, signalling during limb development. This reveals an unexpected specificity of HSPGs in regulating distinct vertebrate Fgfs (Norton, 2005).

The secreted serine protease xHtrA1, expressed in early Xenopus embryos and transcriptionally activated by FGF signals, promotes posterior development in mRNA-injected embryos. xHtrA1 mRNA leads to the induction of secondary tail-like structures, expansion of mesoderm, and formation of ectopic neurons in an FGF-dependent manner. An antisense morpholino oligonucleotide or a neutralizing antibody against xHtrA1 has the opposite effects. xHtrA1 activates FGF/ERK signaling and the transcription of FGF genes. Xenopus Biglycan, Syndecan-4, and Glypican-4 are proteolytic targets of xHtrA1 and heparan sulfate and dermatan sulfate trigger posteriorization, mesoderm induction, and neuronal differentiation via the FGF signaling pathway. The results are consistent with a mechanism by which xHtrA1, through cleaving proteoglycans, releases cell-surface-bound FGF ligands and stimulates long-range FGF signaling (Hou, 2007).

HtrA1 belongs to the HtrA (High temperature requirement-A) family of serine proteases that is well conserved from bacteria to humans. HtrA1 was originally isolated as a gene downregulated in SV40-transformed human fibroblasts. Overexpression of HtrA1 in cancer cells suppresses growth and proliferation in vivo, suggesting that HtrA1 is a candidate tumor suppressor. More recently, a single nucleotide polymorphism in the HtrA1 promoter has been presented as a major risk factor for age-related macular degeneration. HtrA1 binds to and inactivates members of the TGFβ family and modulates insulin-like growth factor (IGF) signals, but its biological function is not yet known (Hou, 2007).

The Xenopus homolog of HtrA1 (xHtrA1) was identified in a direct screen for secreted proteins. xHtrA1 is a modulator of FGF signaling that participates in axial development, mesoderm formation, and neuronal differentiation. xHtrA1 is activated by FGF signals and induces ectopic FGF4 and FGF8 transcription. Biglycan, Syndecan-4, and Glypican-4 are proteolytic targets of xHtrA1; pure heparan sulfate and dermatan sulfate phenocopy xHtrA1 and FGF activities in Xenopus embryos. The results suggest that xHtrA1 acts as a positive regulator of FGF signals and, through proteolytic cleavage of proteoglycans, allows long-range FGF signaling in the extracellular space (Hou, 2007).

Binding of FGF to its receptors, receptor signaling and nuclear FGF

The fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. An understanding of the molecular nature of the interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF-FGFR complex. In this study, based on an exhaustive analysis of the primary sequences of the FGF family, it has been determined that the residues that constitute the primary receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. It is proposed that the FGF-FGFR interaction mediated by the 'conserved' primary site interactions is likely to be similar if not identical for the entire FGF family, whereas the 'variable' secondary sites, on both FGF as well as FGFR mediate specificity of a given FGF to a given FGFR isoform. Furthermore, since the pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, the spatial orientation of the primary receptor-binding site of FGF-2 is found to coincide structurally with the IL-1beta receptor-binding site when the two molecules are superimposed. The structural similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF-FGFR interactions. In the FGF-FGFR model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but also FGF dimerization or oligomerization mediated by HLGAG (Venkataraman, 1999b).

Expression constructs were constructed in which cDNAs encoding individual immunoglobulin-like domains of the keratinocyte growth factor (KGF/FGF-7) receptor were fused to the mouse immunoglobulin heavy chain Fc domain (HFc). The chimera containing immunoglobulin-like domains 2 (D2) and 3 (D3) binds KGF and acidic FGF at high affinities comparable to the native receptor. However, individual immunoglobulin-like domain chimeras demonstrate marked specificity in their ligand interactions. D2-HFc binds acidic FGF at high affinity, whereas it does not detectably interact with KGF. Conversely, D3-HFc binds KGF at high affinity but exhibits no detectable interaction with acidic FGF. Their selective ligand binding properties are confirmed by the specific neutralization of acidic FGF or KGF mitogenic activity using D2 or D3 HFc, respectively. All of these findings establish that the major binding sites for related FGF ligands are localized to distinct receptor immunoglobulin-like domains (Cheon, 1994).

A lipid-anchored Grb2-binding protein links FGF-receptor activation to the Ras/MAPK signaling pathway. Activation of the Ras/MAPK signaling cascade is essential for growth factor-induced cell proliferation and differentiation. A novel protein, designated FRS2 is tyrosine phosphorylated and binds to Grb2/Sos in response to FGF or NGF stimulation. FRS2 is myristylated and this modification is essential for membrane localization, tyrosine phosphorylation, Grb2/Sos recruitment, and MAPK activation. FRS2 functions as a lipid-anchored docking protein that targets signaling molecules to the plasma membrane in response to FGF stimulation to link receptor activation with the MAPK and other signaling pathways essential for cell growth and differentiation. FRS2 is closely related and probably indentical to SNT (suc1-associated neurotrophic factor target), the long-sought target of FGF and NGF receptors (Kouhara, 1997).

The presence of fibroblast growth factor-2 in the nucleus has now been reported both in vitro and in vivo, but its nuclear functions are unknown. Fgf-2 added to nuclear extract binds to protein kinase Ck2 (See Drosophila Casein kinase II ) and nucleolin, a Ck2 natural substrate. Added to baculovirus-infected cell extracts overexpressing Ck2 or its isolated subunits, Fgf-2 binds to the enzyme through its regulatory beta subunit. Using purified proteins, Fgf-2 is shown to directly interact with Ck2 and to stimulate Ck2 activity toward nucleolin. A mitogenic-deficient Fgf-2 mutant protein has an impaired ability to interact with Ck2 and to stimulate Ck2 activity using nucleolin as substrate. It is proposed that in growing cells, one function of nuclear Fgf-2 is to modulate Ck2 activity through binding to its regulatory beta subunit (Bonnet, 1996).

Fibroblast growth factor-2 (FGF-2) immobilized on non-tissue culture plastic promotes adhesion and spreading of bovine and human endothelial cells that are inhibited by anti-FGF-2 antibody. Heat-inactivated FGF-2 retains its cell-adhesive activity despite its incapacity to bind to tyrosine-kinase FGF receptors or to cell-surface heparan sulfate proteoglycans. Recombinant glutathione-S-transferase-FGF-2 chimeras and synthetic FGF-2 fragments identify two cell-adhesive domains in FGF-2 corresponding to amino acid sequences 38-61 and 82-101. Both regions are distinct from the FGF-receptor-binding domain of FGF-2 and contain a DGR sequence that is the inverse of the RGD cell-recognition sequence. Calcium deprivation, RGD-containing eptapeptides, soluble vitronectin (VN), but not fibronectin (FN), inhibit cell adhesion to FGF-2. Conversely, soluble FGF-2 prevents cell adhesion to VN but not FN, thus implicating VN receptor in the cell-adhesive activity of FGF-2. Accordingly, monoclonal and polyclonal anti-alphavbeta3 antibodies prevent cell adhesion to FGF-2. Purified human alphavbeta3 (see Drosophila Myospheroid) binds to immobilized FGF-2 in a cation-dependent manner, and this interaction is competed by soluble VN but not by soluble FN. Anti-alphavbeta3 monoclonal and polyclonal antibodies specifically inhibit mitogenesis and urokinase-type plasminogen activator (uPA) up-regulation induced by free FGF-2 in endothelial cells adherent to tissue culture plastic. These data demonstrate that FGF-2 interacts with alphavbeta3 integrin. This interaction mediates the capacity of the angiogenic growth factor to induce cell adhesion, mitogenesis, and uPA up-regulation in endothelial cells (Rusnati, 1997).

To elucidate the structural determinants governing specificity in fibroblast growth factor (FGF) signaling, the crystal structures were determined for FGF1 and FGF2 complexed with the ligand binding domains (immunoglobulin-like domains 2 [D2] and 3 [D3]) of FGF receptor 1 (FGFR1) and FGFR2, respectively. Highly conserved FGF-D2 and FGF-linker (between D2-D3) interfaces define a general binding site for all FGF-FGFR complexes. Specificity is achieved through interactions between the N-terminal and central regions of FGFs and two loop regions in D3 that are subject to alternative splicing. These structures provide a molecular basis for FGF1 as a universal FGFR ligand and for modulation of FGF-FGFR specificity through primary sequence variations and alternative splicing (Plotnikov, 2000).

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases that can phosphorylate phosphaditylinositides leading to the cell type-specific regulation of intracellular protein kinases. PI3Ks are involved in a wide variety of cellular events including mitogenic signaling, regulation of growth and survival, vesicular trafficking, and control of the cytoskeleton. Some of these enzymes also act downstream of receptor tyrosine kinases or G-protein-coupled receptors. Using two strategies to inhibit PI3K signaling in embryos, the role of PI3Ks during early Xenopus development has been analyzed. A class 1A PI3K catalytic activity is required for the definition of trunk mesoderm during the blastula stages, but is less important for endoderm and prechordal plate mesoderm induction or for organizer formation. It is required in the FGF signaling pathway downstream of Ras and in parallel to the extracellular signal-regulated kinase (ERK) MAP kinases. In addition, ERKs and PI3Ks can synergise to convert ectoderm into mesoderm. These data provide the first evidence that class 1 PI3Ks are required for a specific set of patterning events in vertebrate embryos. Furthermore, they bring new insight into the FGF signaling cascade in Xenopus (Carballada, 2001).

While PI3K has been shown to be required for signal transduction in response to RTK ligands such as PDGF, insulin or EGF, its role in FGF signaling is much less clear cut. On the one hand, several molecules able to bind PI3K subunits, such as dof, act downstream of FGF or are found associated with the FGF receptor. Also, treatment of cultured cell lines with basic FGF can lead to a modest increase in PI3K activity. On the other hand, inhibition of PI3K signaling seldom has a demonstrated direct effect on the response to FGF and in the few cases where this appears to be the case, the role of PI3K is limited to the reorganization of the cytoskeleton or the regulation of exocytosis. In no case has the direct activation by FGF of a target gene been shown to be PI3K dependent. In contrast to the controversial role of PI3K in FGF signaling, activation of the MAP kinase pathway plays a crucial role in FGF signaling. On the basis of the overexpression of activated MAP kinase, it has been suggested that the Ras-dependent activation of this kinase is sufficient to account for the FGF-mediated induction of mesoderm induction and for the direct activation of Xbra (Carballada, 2001).

Using two different strategies to interfere with PI3K signaling, this study provides the first demonstration that PI3K signaling is crucial for the direct activation by FGF of Xbra. PI3K signaling is not involved in the activation of ERK by FGF but rather acts in parallel to the MAP kinase pathway. In contrast to what has been previously proposed, these results thus indicate that, during mesoderm induction, the FGF signaling pathway splits upstream of ERK into at least two cooperating branches. Several questions remain to be addressed. (1) The weak mesoderm induction obtained when both the ERK and PI3K pathways are activated suggests the existence of additional parallel effector pathways downstream of Ras. Several effector pathways, including Ral, Rac/Rho and phospholipase D, have been shown to act downstream of Ras and probably in parallel to ERK and PI3K. It will be important to test the role of these pathways in Xenopus mesoderm formation. It will also be important to position PI3K with respect to laloo, a recently described src-family tyrosine kinase acting in the FGF pathway. (2) PI3K is required for FGF signaling, whether this PI3K activity is modulated by FGF signaling has not been addressed. This could be the case, since in other systems FGF can stimulate, albeit weakly, PI3K activity. In addition, p85 is associated to the FGF receptor in Xenopus embryos during gastrulation. (3) The components acting downstream of PI3K in mesoderm induction must be identified. Several downstream effectors of PI3K have been characterized in cultured cells including GSK3, PKB/Akt, p70 S6k and the GTPases Rac and Rho. The results presented here do not support a role for GSK3 downstream of PI3K in early embryos, since expression of Siamois, a direct target of the beta-catenin/GSK3 pathway, is not affected by treatment with LY294002. It will be interesting to test a potential role for PKB/Akt and Rac/Rho in mesoderm induction downstream of PI3K. The availability of constitutively active or dominant negative forms of proteins acting in the Ras and PI3K pathways in other systems, coupled with the convenience of the Xenopus system, will help shed light on these issues (Carballada, 2001).

In developing limbs, numerous signaling molecules have been identified but less is known about the mechanisms by which such signals direct patterning. Signal transduction pathways in the chicken limb bud have been explored. A cDNA encoding RACK1, a protein that binds and stabilizes activated protein kinase C (PKC), was isolated in a screen for genes induced by retinoic acid (RA) in the chick wing bud. Fibroblast growth factor (FGF) also induces RACK1 and such induction of RACK1 expression is accompanied by a significant augmentation in the number of active PKC molecules and an elevation of PKC enzymatic activity. This suggests that PKCs mediate signal transduction in the limb bud. Application of chelerythrine, a potent PKC inhibitor, to the presumptive wing region results in buds that do not express sonic hedgehog (Shh) and develop into wings that are severely truncated. This observation suggests that the expression of Shh depends on PKCs. Providing ectopic SHH protein, RA or ZPA grafts overcomes the effects of blocking PKC with chelerythrine and results in a rescue of the wing morphology. Taken together, these findings suggest that the responsiveness of Shh to FGF is mediated, at least in part, by PKCs (Lu, 2001).

Intracelluar functions of FGF

Fibroblast growth factor-1 (FGF-1) has both extra- and intracellular functions. To identify intracellular binding partners for FGF-1, proteins were isolated from U2OS human osteosarcoma cells interacting specifically with FGF-1. One of the isolated proteins was identified as protein kinase CK2 (CK2). Evidence is provided that FGF-1 binds to both the catalytic alpha-subunit and to the regulatory ß-subunit of CK2. The interaction between FGF-1 and CK2^a and ß was characterized by surface plasmon resonance, giving KD values of 0.4 ± 0.3 and 1.2 ± 0.2 microM, respectively. By using a novel assay for intracellular protein interaction, FGF-1 and CK^a were shown to interact in vivo. In vitro, FGF-1 and FGF-2 are phosphorylated by CK^a, and the presence of FGF-1 or FGF-2 was found to enhance the autophosphorylation of CK2ß. A correlation between the mitogenic potential of FGF-1 mutants and their ability to bind to CK^a was observed (Skjerpen, 2002).

Antagonism of FGF signaling by Sprouty homologs

SPRY2, a human homolog of Drosophila Sprouty, is involved FGF2 signaling. In primary human dermal endothelial cells (MVEC) SPRY2 mRNA is transiently upregulated in response to FGF2. Overexpression of SPRY2 in A375 cells leads to the secretion of a soluble factor that inhibits FGF2- but not VEGF-stimulated proliferation of MVEC. Direct administration of recombinant SPRY2 protein has no effect on MVEC proliferation. However, SPRY2 protein binds the intracellular adaptor protein GRB2, indicating an intracellular localization. A SPRY2/GFP fusion protein remains in the cell, further supporting the intracellular localization of SPRY2. So the intracellular protein SPRY2 is involved in the non-cell autonomous inhibitory effect indirectly, via regulating the secretion of an inhibitor of FGF2 signaling in vertebrates, the evidence of which is presented here for the first time (Glienke, 2000).

Regulation of FGF signaling

The importance of endogenous antagonists in intracellular signal transduction pathways is becoming increasingly recognized. There is evidence in cultured mammalian cells that Pyst1/MKP3, a dual specificity protein phosphatase, specifically binds to and inactivates ERK1/2 mitogen-activated protein kinases (MAPKs). High-level Pyst1/Mkp3 expression has recently been found at many sites of known FGF signaling in mouse embryos, but the significance of this association and its function are not known. High-level expression of Chicken Pyst1/Mkp3 in neural plate correlates with active MAPK. FGF signaling regulates Pyst1 expression in developing neural plate and limb bud by ablating and/or transplanting tissue sources of FGFs and by applying FGF protein or a specific FGFR inhibitor (SU5402). by applying a specific MAP kinase kinase inhibitor (PD184352) it has been shown that Pyst1 expression is regulated via the MAPK cascade. Overexpression of Pyst1 in chick embryos reduces levels of activated MAPK in neural plate and alters its morphology and retards limb bud outgrowth. It is concluded that Pyst1 is an inducible antagonist of FGF signaling in embryos and acts in a negative feedback loop to regulate the activity of MAPK. These results demonstrate both the importance of MAPK signaling in neural induction and limb bud outgrowth and the critical role played by dual specificity MAP kinase phosphatases in regulating developmental outcomes in vertebrates (Eblaghie, 2003).

Invertebrate FGFs

The proper guidance of the C. elegans hermaphrodite sex myoblasts (SMs) requires the genes egl-15 and egl-17. egl-15 has been shown to encode the C. elegans orthologue of the fibroblast growth factor receptor. egl-17 was cloned and is a member of the fibroblast growth factor family, one of the first functional invertebrate FGFs known. egl-17 shares homology with other FGF members, conserving the key residues required to form the distinctive tertiary structure common to FGFs. The SM migration defect seen in egl-17 mutant animals represents complete loss of egl-17 function. While mutations in egl-17 affect only SM migrations, mutations in egl-15 can result in larval arrest, and scrawny body morphology (Burdine, 1997).

During the development of the egg-laying system in Caenorhabditis elegans hermaphrodites, central gonadal cells organize the alignment of the vulva with the sex myoblasts, the progenitors of the egg-laying muscles. A fibroblast growth factor [EGL-17(FGF)] and an FGF receptor [EGL-15(FGFR)] are involved in the gonadal signals that guide the migrations of the sex myoblasts (SMs). SMs are generated in the posterior half of the mid-body region and undergo anterior migrations to final positions that flank the center of the developing gonad. When the gonad is destroyed by laser ablation, the SMs still migrate anteriorly but take up final positions within a broad, centrally dispersed range. Thus, the gonad is responsible for the precise positioning of the SMs. This precise positioning appears to result from a gonad-dependent attractive signaling mechanism, since the SMs can migrate into novel territory in response to an altered positioning of the gonad. EGL-17(FGF) can act as an instructive guidance cue to direct the sex myoblasts to their final destinations. egl-17 reporter constructs are expressed in the primary vulval cell and EGL-17(FGF) expression in this cell correlates with the precise positioning of the sex myoblasts. It is postulated that EGL-17(FGF) helps to coordinate the development of a functional egg-laying system, linking vulval induction with proper sex myoblast migration. Vulval expression of egl-17 is dependent on vulval induction, requiring the action of a Ras-MAP kinase signal that is activated by Epidermal growth factor dependent signaling. egl-17 is expressed in vulval cells early in primary cell lineages and late in secondary cell lineages. A gonad-dependent repulsion mechanism may occur when egl-17 dependent function is eliminated (Burdine, 1998).

Growth factors and morphogens need to be secreted to act on distant cells during development and in response to injury. Evidence is presented that efficient export of a fibroblast growth factor (FGF), EGL-17, from the C. elegans developing vulva requires the lipoprotein receptor-related proteins Ce-LRP-1 and Ce-LRP-2 and a cytoplasmic adaptor protein, Ce-DAB-1 (Disabled). Lipoprotein receptors are transmembrane proteins best known for their roles in endocytosis. Ce-LRP-1 and Ce-LRP-2 possess a conserved intraluminal domain that can bind to EGL-17, as well as a cytosolic FXNPXY motif that can bind to Ce-DAB-1. Ce-DAB-1 contains signals that confer subcellular localization to Golgi-proximal vesicles. These results suggest a model in which Ce-DAB-1 coordinates selection of receptors and cargo, including EGL-17, for transport through the secretory pathway (Kamikura, 2003).

Dab family adaptor proteins interact functionally with lipoprotein receptors in both nematodes and mammals, even though the biological processes they mediate vary greatly. Ce-DAB-1 regulates secretion, Dab2 regulates endocytosis in the kidney, and Dab1 relays extracellular signals during brain development, each via lipoprotein receptors. Although the role of Ce-DAB-1 in signaling is unclear, the high degree of functional conservation across species suggests that vertebrate Dab family members or other PTB-containing proteins may participate in regulated traffic of lipoprotein receptors and associated cargoes to the cell surface. Indeed, it is possible that an early embryonic requirement for Dab2 might be a consequence of altered protein traffic in polarized epithelial cells of the embryo (Kamikura, 2003).

Although many molecules are necessary for neuronal cell migrations in C. elegans, no guidance cues are known to be essential for any of these cells to migrate along the anteroposterior (AP) axis. The fibroblast growth factor (FGF) EGL-17, an attractant for the migrating sex myoblasts (SMs), repels the CANs, a pair of neurons that migrate posteriorly from the head to the center of the embryo. Although mutations in genes encoding EGL-17/FGF and a specific isoform of its receptor EGL-15/FGFR had little effect on CAN migration -- they enhanced the CAN migration defects caused by mutations in other genes. Two cells at the anterior end of the embryo express EGL-17/FGF, raising the possibility that EGL-17/FGF functions as a repellent for migrating CANs. Consistent with this hypothesis, ectopic expression of EGL-17/FGF shifted the final CAN cell positions away from these novel sites of expression. Cell-specific rescue experiments demonstrated that EGL-15/FGFR acts in the CANs to promote their migration. The tyrosine phosphatase receptor CLR-1 regulates CAN migration by inhibiting EGL-15/FGFR signaling, and the FGFR adaptor protein SEM-5/GRB2 may mediate EGL-15/FGFR signaling in CAN migration. Thus, EGL-17/FGF signaling through an EGL-15/FGFR isoform and possibly SEM-5/GRB2 mediates both attraction of the SMs and repulsion of the CANs. This study also raises the possibility that several guidance cues regulate cell migrations along the C. elegans AP axis, and their role in these migrations may only be revealed in sensitized genetic backgrounds (Fleming, 2005).

In C. elegans, the gene vab-8 is both necessary and sufficient for posteriorly directed migrations of cells and growth cones. Most posterior migrations require vab-8, and ectopic expression of vab-8 can reroute anteriorly projected axons towards the posterior. The vab-8 locus encodes at least two novel intracellular proteins that act in the cells to promote their migration. How these proteins regulate these migrations, however, remains unknown (Fleming, 2005).

Unlike VAB-8, the C. elegans transmembrane protein MIG-13 plays a nonautonomous role in guiding cell migrations along the AP axis. mig-13 loss-of-function alleles display more specific defects than vab-8 mutants, disrupting the anterior directed migrations of only the BDU neurons and descendants of the right Q neuroblast. Ectopic expression of mig-13 from a heat shock promoter, however, induces an anterior shift in the final positions of neurons that migrate in either direction along the AP axis, indicating that MIG-13 plays a broader role than was suggested by the effects of mig-13 mutants. As with VAB-8, the role of MIG-13 in these migrations remains unclear (Fleming, 2005).

In C. elegans, the fibroblast growth factor (FGF) homolog EGL-17 functions as an attractant for the precise positioning of the anteriorly directed migrations of the sex myoblasts (SMs). In early larval stages, the SMs migrate from the posterior midbody to positions flanking the center of the gonad. During SM migration, EGL-17 is expressed in the primary vulval precursor cells (VPCs) and the dorsal uterine (DU) cells of the somatic gonad, which define the final destination of the SMs. EGL-17 signals through the FGF receptor (FGFR) EGL-15 to attract SMs (Fleming, 2005).

In an effort to understand AP guidance in C. elegans, focus was placed on the posterior migrations of the CANs, a pair of bilaterally symmetric neurons that are born in the head and migrate to the middle of the embryo. Although a previous screen for CAN migration mutants identified a number of genes, none of them encoded guidance cues. One explanation for this outcome is that multiple cues contribute to CAN migration, and therefore removing one might result in only subtle CAN migration defects. To test this hypothesis, sensitized genetic backgrounds, specifically a vab-8 mutation was used to re-evaluate the potential role of secreted molecules in CAN migration. The use of these sensitized backgrounds revealed a role for FGF in CAN migration (Fleming, 2005).

Striated muscles from Drosophila and several vertebrates extend plasma membrane to facilitate the formation of the neuromuscular junction (NMJ) during development. However, the regulation of these membrane extensions is poorly understood. In C. elegans, the body wall muscles (BWMs) also have plasma membrane extensions called muscle arms that are guided to the motor axons where they form the postsynaptic element of the NMJ. To investigate the regulation of muscle membrane extension, 871 genes were screened by RNAi for ectopic muscle membrane extensions (EMEs) in C. elegans. An FGF pathway, including let-756(FGF), egl-15(FGF receptor), sem-5(GRB2) and other genes were found to negatively regulate plasma membrane extension from muscles. Although compromised FGF pathway activity results in EMEs, hyperactivity of the pathway disrupts larval muscle arm extension, a phenotype called muscle arm extension defective or MAD. Expression of egl-15 and sem-5 in the BWMs are each necessary and sufficient to prevent EMEs. Furthermore, let-756 expression from any one of several tissues can rescue the EMEs of let-756 mutants, suggesting that LET-756 does not guide muscle membrane extensions. The screen also revealed that loss-of-function in laminin and integrin components results in both MADs and EMEs, the latter of which are suppressed by hyperactive FGF signaling. These data are consistent with a model in which integrins and laminins are needed for directed muscle arm extension to the nerve cords, while FGF signaling provides a general mechanism to regulate muscle membrane extension (Dixon, 2006).

Two axial structures, a neural tube and a notochord, are key structures in the chordate body plan, and a closer look at these structures furthers understanding of the origin of chordates. The neural tube of ascidian larvae is composed of about 340 cells, and is divided into three regions along the anteroposterior axis, which are, from anterior to posterior, the sensory vesicle, the visceral ganglion and the caudal neural tube. The sensory vesicle is composed solely of the a-line (anterior-animal) cells. The visceral ganglion present at the junction between the trunk and tail consists of the A-line (anterior-vegetal) cells. The caudal neural tube running along the length of the tail consists of four (dorsal, ventral and two lateral) rows of ependymal cells: the lateral and ventral cells are of A-line origin and the dorsal cells are of b-line (posterior-animal) origin. Beneath the neural tube, a stack of exactly 40 notochord cells runs along the tail. The anterior 32 cells (primary notochord) and the posterior 8 cells (secondary notochord) are derived from A-line and b-line cells, respectively. To expand knowledge on mechanisms of development of the neural tube in lower chordates, isolation and characterization of HrzicN, a new member of the Zic family gene of the ascidian, Halocynthia roretzi, was undertaken. HrzicN expression is detected by whole-mount in situ hybridization in all neural tube precursors, all notochord precursors, anterior mesenchyme precursors and a part of the primary muscle precursors. Expression of HrzicN in a- and b-line neural tube precursors was detected from early gastrula stage to the neural plate stage, while expression in other lineages is observed between the 32-cell and the 110-cell stages. HrzicN function was investigated by disturbing translation using a morpholino antisense oligonucleotide. Embryos injected with HrzicN morpholino ('HrzicN knockdown embryos') exhibit failure of neurulation and tail elongation, and develop into larvae without a neural tube and notochord. Analysis of neural marker gene expression in HrzicN knockdown embryos has revealed that HrzicN plays critical roles in distinct steps of neural tube formation in the a-line- and A-line-precursors. In particular HrzicN is required for early specification of the neural tube fate in A-line precursors. Involvement of HrzicN in the neural tube development was also suggested by an overexpression experiment. However, analysis of mesodermal marker gene expression in HrzicN knockdown embryos revealed unexpected roles for this gene in the development of mesodermal tissues. Regulation of HrzicN expression by FGF-like signaling was investigated. This signaling has been shown to be involved in induction of the a- and b-line neural tube, the notochord and the mesenchyme cells in Halocynthia embryos. Using an inhibitor of FGF-like signaling, it has been shown that HrzicN expression in the a- and b-line neural tube, but not in the A-line lineage and mesodermal lineage, depends on FGF-like signaling. Based on these data, roles of HrzicN as a key gene in the development of the neural tube and the notochord are discussed (Wada, 2002).

Ascidian larvae develop mesenchyme cells in their trunk. A fibroblast growth factor (FGF9/16/20) is essential and sufficient for induction of the mesenchyme in Ciona savignyi. Two basic helix-loop-helix (bHLH) genes named Twist-like1 and Twist-like2 have been identified as downstream factors of this FGF. These two genes are phylogenetically closely related to each other, and are expressed specifically in the mesenchymal cells after the 110-cell stage. Gene-knockdown experiments using a specific morpholino oligonucleotide demonstrate that Twist-like1 plays an essential role in determination of the mesenchyme and that Twist-like2 is a downstream factor of Twist-like1. In addition, both overexpression and misexpression of Twist-like1 converts non-mesenchymal cells to mesenchymal cells. The upstream regulatory mechanisms of Twist-like1 are different between B-line mesenchymal cells and the A-line mesenchymal cells called 'trunk lateral cells'. FGF9/16/20 is required for the expression of Twist-like1 in B-line mesenchymal precursor cells, whereas FGF, FoxD and another novel bHLH factor called NoTrlc (for No Trunk lateral cells, because it is essential for the differentiation of TLCs, which give rise to blood cells and body-wall muscles after metamorphosis) are required for Twist-like1 to be expressed in the A-line mesenchymal precursor cells. Therefore, two different but partially overlapping mechanisms are required for the expression of Twist-like1 in the mesenchymal precursors, that triggers the differentiation of the mesenchyme in Ciona embryos (Imai, 2003).

Fgf mutation

Fibroblast growth factor-6 (FGF-6) belongs to a family of cytokines that control cell proliferation, cell differentiation, and morphogenetic events. Individual FGFs are either expressed widely or in a restricted pattern during embryonic, fetal, and adult life. FGF-6 exhibits a restricted expression profile predominantly in the myogenic lineage. Important functions in wound healing and tissue regeneration have been proposed for various FGFs in the past, although data from knockout mice have not supported this view. The FGF-6 gene was inactivated in mice to investigate the role of FGF-6 in skeletal muscle development and regeneration. Wild-type mice up-regulate FGF-6 after skeletal muscle injuries and completely restore experimentally damaged skeletal muscle. In contrast, FGF-6(-/-) mutant mice show a severe regeneration defect with fibrosis and myotube degeneration. The number of MyoD- and Myogenin-expressing activated satellite cells after injury are significantly reduced in mutants. This reduction is not caused by a reduced pool of quiescent satellite cells but presumably by a lack of activation or proliferation. Interbreeding of FGF-6(-/-) mutants with mdx mice, which lack Dystrophin (thought to be required for the stabilization of the sarcolemma), leads to striking dystrophic changes in skeletal muscles of double homozygous mice characterized by myotube degeneration, the presence of large amounts of mononuclear cells, and deposition of collagen. RNA analysis reveals an up-regulation of MyoD mRNA in mdx but not in FGF-6(-/-)/mdx double mutant mice. It is concluded that FGF-6 is a critical component of the muscle regeneration machinery in mammals, possibly by stimulating or activating satellite cells (Floss, 1997).

Mammalian palatogenesis depends on interactions between the stomodium-derived epithelium and the cranial neural crest-derived ectomesenchyme. Fibroblast growth factor 10 (FGF10) is a mesenchymal signaling factor that guides the morphogenesis of multiple organs through tissue-tissue interactions. This is consistent with widespread agenesis and dysgenesis of organs observed in Fgf10^−/− mice. A wide-open cleft secondary palate is present in Fgf10 homozygous null mutant mice. Fgf10 transcripts are detected in the palatal mesenchyme from E11.5 to E13.5 during normal palatogenesis and are enriched in the anterior and middle portions of the palatal shelves. In Fgf10^−/− embryos, histological analyses revealed aberrant adhesion of the palatal shelves with the tongue in the anterior and fusion with the mandible in the middle and posterior beginning at E13.5, which could prevent normal elevation of the palatal shelves leading to a cleft palate. TUNEL and BrdU assays demonstrate significant levels of apoptosis in the medial edge epithelium (MEE) but unaltered cell proliferation in mutant palatal shelves. At the molecular level, Fgf10 is shown to be epistatic to Jagged2 and Tgfβ3 in the developing palate. Notably, the expression of Jagged2 is downregulated throughout the palate epithelium in Fgf10 mutants while Tgfβ3 is misexpressed in the palatal epithelium at the oral side. These results demonstrate that mesenchymally expressed Fgf10 is necessary for the survival of MEE cells and for the normal expression of Jagged2 and Tgfβ3 in the palatal epithelium during mammalian palatogenesis (Alappat, 2004).

FGF receptor during oocyte maturation

FGF signaling is critical for establishing the Xenopus laevis embryonic body plan and requires the expression of functional FGF receptor during early embryogenesis. FGF receptor-1 (XFGFR) maternal mRNA is present in immature oocytes, but the protein is not expressed until oocyte maturation. Endogenous XFGFR translation begins just prior to germinal vesicle breakdown (GVBD) and translation depends on completion of earlier meiotic events. XFGR translation is undetectable in immatue oocytes and for several hours following progesterone addition. Six hours after progersterone addition, while 5% of the oocytes have undergone GVBD, a very low level of XFGFR protein is detected in oocytes that have not undergone GVBD at that time. Nine hours after progesterone addtion (1 h prior to GVBD50) a significant level of XFGFR translation is observed; this rate of translation is maintained until at least 5 hr after GVBD50. While control oocytes mature, and accumulate maximum levels of XFGFR protein, c-mos ablated oocytes do not mature, and extracts of these oocytes are devoid of XFGFR protein. These results indicate that SFGFR translation is activated just prior to GVBD and is dependent on the completion of upstream meiotic cell cycle events that are triggered by the activity of c-mos. The previously identified XFGFR 3'UTR translation inhibitory element (TIE), which is necessary and sufficient for repressing translation in the immature oocyte, also regulates the onset of translation during oocyte maturation. In addition, cytoplasmic polyadenylation of XFGFR RNA is regulated independently of TIE-mediated translation and is not sufficient to activate the translation of XFGFR. These experiments reveal that polyadenylation and translational activation are separable events in this mRNA, each of which is timed and regulated independently (Culp, 1998).

Table of contents

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

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