Interactive Fly, Drosophila

FGF and kidney morphogenesis

The importance of proportioning kidney size to body volume has been established by clinical studies that demonstrate that in-born defecits of nephron number predispose the kidney to disease. As the kidney develops, the expanding ureteric bud or renal collecting system induces surrounding metanephric mesenchyme to proliferate and differentiate into nephrons. Thus, it is likely that nephron number is related to ureteric bud growth. The expression patterns of mRNAs encoding fibroblast growth factor-7 (FGF-7) and its high affinity receptor suggested that FGF-7 signaling may play a role in regulating ureteric bud growth. To test this hypothesis, kidneys from FGF-7-null and wild-type mice were examined. Results of these studies demonstrate that the developing ureteric bud and mature collecting system of FGF-7-null kidneys is markedly smaller than in wild type. Furthermore, morphometric analyses indicate that mature FGF-7-null kidneys have 30+/-6% fewer nephrons than wild-type kidneys. In vitro experiments demonstrate that elevated levels of FGF-7 augment ureteric bud growth and increase the number of nephrons that form in rodent metanephric kidney organ cultures. Collectively, these results demonstrate that FGF-7 levels modulate the extent of ureteric bud growth during development and the number of nephrons that eventually form in the kidney (Qiao, 1999).

The kidney of the Gpc3-/- mouse, a novel model of human renal dysplasia, is characterized by selective degeneration of medullary collecting ducts preceded by enhanced cell proliferation and overgrowth during branching morphogenesis. Cellular and molecular mechanisms underlying this renal dysplasia have been identified. Glypican-3 (GPC3) deficiency is associated with abnormal and contrasting rates of proliferation and apoptosis in cortical (CCD) and medullary collecting duct (MCD) cells. In CCD, cell proliferation is increased threefold. In MCD, apoptosis was increased 16-fold. Expression of Gpc3 mRNA in ureteric bud and collecting duct cells suggests that GPC3 can exert direct effects in these cells. Indeed, GPC3 deficiency abrogates the inhibitory activity of BMP2 on branch formation in embryonic kidney explants, converts BMP7-dependent inhibition to stimulation, and enhances the stimulatory effects of KGF (FGF-7). Similar comparative differences are found in collecting duct cell lines derived from GPC3-deficient and wild type mice and induced to form tubular progenitors in vitro, suggesting that GPC3 directly controls collecting duct cell responses. It is proposed that GPC3 modulates the actions of stimulatory and inhibitory growth factors during branching morphogenesis (Grisaru, 2001).

The molecular basis for observations in ureteric bud and collecting duct cells remains to be determined. The demonstration that bFGF forms a molecular complex with cell surface heparan sulfate and the FGF cell surface receptor suggests that GPC3 may physically interact with receptors that bind BMP2, BMP7, and KGF. The opposite response of collecting duct cells to GPC3 deficiency, that is, inhibition of BMP2 activity and enhancement of KGF activity, suggests that the consequences of these interactions may differ. A second possibility is that GPC3 may function via independent signaling pathways that physically interact at the postreceptor level with BMP and KGF signaling intermediates. Alternatively, the GPC3 and growth factor-signaling pathways may interact indirectly by regulating competing or complementary gene products. Increasing evidence regarding the nature of inhibitory and stimulatory BMP-dependent signaling pathways in collecting duct cells provides a basis to determine the nature of GPC3 interactions with BMP2 and BMP7 (Grisaru, 2001).

Together with glial-derived neurotrophic factor (GDNF), soluble factors present in a metanephric mesenchyme (MM) cell conditioned medium (BSN-CM) are necessary to induce branching morphogenesis of the isolated ureteric bud (UB) in vitro. Fibroblast growth factors (FGFs) modulated this process. RT-PCR has revealed the expression of two FGF receptors [FGFR1(IIIc) and FGFR2(IIIb) in isolated embryonic day 13 rat UBs] which by indirect immunofluorescence display a uniform distribution. Rat kidney organ culture experiments in the presence of a soluble FGFR2(IIIb) chimera or a neutralizing antibody to FGF7 suggest an important contribution of FGFs other than FGF7 to the branching program. Several FGFs, including FGF1, FGF2, FGF7 and FGF10, in combination with GDNF and BSN-CM affect growth and branching of the isolated UB, albeit with very different effects. FGF1 and FGF7 are at extreme ends of the spectrum, with FGF10 (more FGF1-like) and FGF2 (more FGF7-like) falling in between. FGF1 induces the formation of elongated UB branching stalks with distinct proliferative ampullary tips, whereas FGF7 induces amorphous buds displaying nonselective proliferation with little distinction between stalks and ampullae. FGF1 treatment induces cytoskeletal organization, intercellular junctions and lumens along the stalk portion of the developing tubules, while the ampullary regions contains 'less differentiated' cells with an abundant secretory apparatus. In contrast, FGF7-induced UBs display this 'less differentiated' morphology regardless of position on the structure and are virtually indistinguishable from FGF1-induced ampullae. Consistent with this, FGF7-induced UBs express more markers of cell proliferation than do FGF1-induced UBs; FGF1 causes the UB to express cytoskeletal proteins, extracellular matrix proteins, and at least one integrin, some of which may be important in UB branch elongation. Thus, while the various FGFs examined all support UB growth, FGF1 and FGF10 appear to be more important for branching and branch elongation, and may thus play a role in determination of nephron number and patterning in the developing kidney. These in vitro data may help to explain results from knockout and transgenic studies and suggest how different FGFs may, together with GDNF and other factor(s) secreted by MM cells, regulate branching morphogenesis of the UB by their relative effects on its growth, branching and branch elongation and differentiation, thereby affecting patterning in the developing kidney (Qiao, 2002).

FGF and pancreatic development

The development of the pancreas depends on epithelial-mesenchymal interactions. Fibroblast growth factors (FGFs) and their receptors (FGFRs 1-4) have been identified as mediators of epithelial-mesenchymal interactions in different organs. FGFR-2 IIIb and its ligands FGF-1, FGF-7, and FGF-10 are expressed throughout pancreatic development. In mesenchyme-free cultures of embryonic pancreatic epithelium, FGF-1, FGF-7, and FGF-10 stimulate the growth, morphogenesis, and cytodifferentiation of the exocrine cells of the pancreas. The role of FGFs signaling through FGFR-2 IIIb was further investigated by inhibiting FGFR-2 IIIb signaling in organocultures of pancreatic explants (epithelium + mesenchyme) by using either antisense FGFR-2 IIIb oligonucleotides or a soluble recombinant FGFR-2 IIIb protein. Abrogation of FGFR-2 IIIb signaling resulted in a considerable reduction in the size of the explants and in a 2-fold reduction of the development of the exocrine cells. These results demonstrate that FGFs signaling through FGFR-2 IIIb play an important role in the development of the exocrine pancreas (Miralles, 1999).

Fgf10 plays an essential role in mesenchymal-epithelial interactions during the development of the pancreas. Fgf10 is expressed in the mesenchyme directly adjacent to the early dorsal and ventral pancreatic epithelial buds. In Fgf10-/- mouse embryos, the evagination of the epithelium and the initial formation of the dorsal and ventral buds appear normal. However, the subsequent growth, differentiation and branching morphogenesis of the pancreatic epithelium are arrested; this is primarily due to a dramatic reduction in the proliferation of the epithelial progenitor cells marked by the production of the homeobox protein PDX1. Furthermore, FGF10 restores the population of PDX1-positive cells in organ cultures derived from Fgf10-/- embryos. These results indicate that Fgf10 signaling is required for the normal development of the pancreas and should prove useful in devising methods to expand pancreatic progenitor cells (Bhushan, 2001).

FGF and posterior mesoderm development in zebrafish

Fibroblast growth factor (Fgf) signaling plays an important role during development of posterior mesoderm in vertebrate embryos. Blocking Fgf signaling by expressing a dominant-negative Fgf receptor inhibits posterior mesoderm development. In mice, Fgf8 appears to be the principal ligand required for mesodermal development; mouse Fgf8 mutants do not form mesoderm. In zebrafish, Fgf8 is encoded by the acerebellar locus, and, similar to its mouse otholog, is expressed in early mesodermal precursors during gastrulation. However, zebrafish fgf8 mutants have only mild defects in posterior mesodermal development, suggesting that it is not the only Fgf ligand involved in the development of this tissue. An fgf8-related gene has been identified in zebrafish, fgf24, that is co-expressed with fgf8 in mesodermal precursors during gastrulation. Using morpholino-based gene inactivation, the function of fgf24 during development was analyzed. Inhibiting fgf24 function alone has no affect on the formation of posterior mesoderm. Conversely, inhibiting fgf24 function in embryos mutant for fgf8 blocks the formation of most posterior mesoderm. Thus, fgf8 and fgf24 are together required to promote posterior mesodermal development. Phenotypic and genetic evidence is provided that both these Fgf signaling components interact with no tail and spadetail, two zebrafish T-box transcription factors that are required for the development of all posterior mesoderm. Last, fgf24 is shown to be expressed in early fin bud mesenchyme; inhibiting fgf24 function results in viable fish that lack pectoral fins (Draper, 2003).

FGF, myogenesis and tendon development

Development of the myotome within somites depends on unknown signals from the neural tube. The present study tested the ability of basic fibroblast growth factor (bFGF), transforming growth factor-beta1 (TGF-beta1) and TGF-beta family member dorsalin-1 (dsl-1) to promote myogenesis in stage 10-14 chick paraxial mesoderm utilizing 72 hour explant cultures. Each of these factors alone and the combination of bFGF with dsl-1 has little or no myogenic-promoting activity, but the combination of bFGF with TGF-beta1 demonstrates a potent dose-dependent effect. bFGF also enhances the survival/proliferation of somite cells. 98% of stage 10-11 caudal segmental plate explants treated with bFGF plus TGF-beta1, exhibit myosin heavy chain (MHC)-positive cells (avg.=60 per explant), whereas only 15% of similarly treated somites respond with an average of 5 MHC-positive cells. Thus at stage 10-11, there are rostrocaudal differences in myogenic responsiveness with the caudal (more 'immature') paraxial mesoderm being more myogenically responsive to these factors than are somites. 17% of stage 10-11 caudal segmental plate explants exhibit several MHC-positive cells even when cultured without added growth factors, further demonstrating a different myogenic potential of the caudal paraxial mesoderm. Stage 13-14 paraxial mesoderm also exhibits a myogenic response to bFGF/TGF-beta1 but, unlike stage 10-11 embryos, both somites and segmental plate exhibit a strong response. A two-step mechanism for the bFGF/TGF-beta1 effect is suggested by the finding that only TGF-beta1 is required during the first 12 hours of culture, whereas bFGF plus a TGF-beta-like factor are required for the remainder of the culture. The biological relevance of the findings with bFGF is underscored by the observation that a monoclonal antibody to bFGF inhibits myogenic signaling from the dorsal neural tube. However, a monoclonal antibody that can neutralize the three factors TGF-beta1, TGF-beta2 and TGF-beta3 does not block myogenic signals from the neural tube, raising the possibility that another TGF-beta family member may be involved in vivo (Stern, 1997).

In vitro somite myogenesis is regulated by neural tube and notochord factors including Wnt, Sonic hedgehog (Shh), and basic fibroblast growth factor (bFGF) together with transforming growth factor-beta1 (TGF-beta1). Insulin and insulin-like growth factors I and II (IGF-I and -II) also promote myogenesis in explant cultures containing single somites or somite-sized pieces of segmental plate mesoderm from 2-day (stage 10-14) chicken embryos. The combination of insulin/IGFs with bFGF plus TGF-beta1 promotes even higher levels of myogenesis. Shh promotes myogenesis in this in vitro system, and Shh interacts synergistically with insulin/IGFs to promote high levels of myogenesis. RT-PCR analysis has detected insulin, IGF-II, insulin receptor, and IGF receptor mRNAs in both the neural tube and the somites, whereas IGF-I transcripts are detected in entire embryos but not in the neural tube or somites. Treatment of somite-neural tube cocultures with anti-insulin, anti-IGF-II, anti-insulin receptor, or anti-IGF receptor blocking antibodies causes a significant decrease in myogenesis. These results are consistent with the hypothesis that systemic IGF-I as well as insulin and IGF-II secreted by the neural tube act as additional early myogenic signals during embryogenesis. Further studies indicate that insulin, IGFs, bFGF, and Shh also stimulate somite cell proliferation and influence apoptosis (Pirskanen, 2000).

The Fgf4 gene encodes an important signaling molecule that is expressed in specific developmental stages, including the inner cell mass of the blastocyst, the myotomes, and the limb bud apical ectodermal ridge (AER). Using a transgenic approach, overlapping but distinct enhancer elements have been identified in the Fgf4 3' untranslated region necessary and sufficient for myotome and AER expression. The hypothesis that Fgf4 is a target of myogenic bHLH factors has been investigated in this study. By mutational analysis it has been shown that a conserved E box located in the Fgf4 myotome enhancer is required for Fgf4-lacZ expression in the myotomes. A DNA probe containing the E box binds MYF5, MYOD, and bHLH-like activities from nuclear extracts of differentiating C2-7 myoblast cells, and both MYF5 and MYOD can activate gene expression of reporter plasmids containing the E-box element. Analyses of Myf5 and MyoD knockout mice harboring Fgf4-lacZ transgenes show that Myf5 is required for Fgf4 expression in the myotomes, while MyoD is not, but MyoD can sustain Fgf4 expression in the ventral myotomes in the absence of Myf5. Sonic hedgehog (Shh) signaling has been shown to have an essential inductive function in the expression of Myf5 and MyoD in the epaxial myotomes, but not in the hypaxial myotomes. Expression of an Fgf4-lacZ transgene in Shh-/- embryos is suppressed not only in the epaxial but also in the hypaxial myotomes, while it is maintained in the AER. This suggests that Shh mediates Fgf4 activation in the myotomes through mechanisms independent of its role in the activation of myogenic factors. Thus, a cascade of events, involving Shh and bHLH factors, is responsible for activating Fgf4 expression in the myotomes in a spatial- and temporal-specific manner (Fraidenraich, 2000).

The community effect is an interaction among a group of many nearby precursor cells, necessary for them to maintain tissue-specific gene expression and differentiate co-ordinately. During Xenopus myogenesis, the muscle precursor cells must be in group contact throughout gastrulation in order to develop into terminally differentiated muscle. The molecular basis of this community interaction has not to date been elucidated. An assay has been developed for testing potential community factors, in which isolated muscle precursor cells are treated with a candidate protein and cultured in dispersion. A number of candidate factors have been tested, and only eFGF protein is able to mediate a community effect, stimulating stable muscle-specific gene expression in demonstrably single muscle precursor cells. In contrast, Xwnt8, bFGF, BMP4 and TGFbeta2 do not show this capacity. eFGF is expressed in the muscle precursor cells at the right time to mediate the community effect. Moreover, the time when the muscle precursor cells are sensitive to eFGF corresponds to the period of the endogenous community effect. Finally, FGF signaling is shown to be essential for endogenous community interactions. It is concluded that eFGF is likely to mediate the community effect in Xenopus myogenesis (Standley, 2001).

This paper addresses the molecular mechanisms that regulate the transcriptional activation of the myogenic regulatory factor XmyoD in the skeletal muscle lineage of Xenopus laevis. Using antisense morpholino oligonucleotide-mediated inhibition, it has been shown that the signaling molecule embryonic fibroblast growth factor (eFGF), which is the amphibian homolog of FGF4, is necessary for the initial activation of XmyoD transcription in myogenic cells. eFGF can activate the expression of XmyoD in the absence of protein synthesis, indicating that this regulation is direct. These data suggest that regulation of XmyoD expression may involve a labile transcriptional repressor. In addition, eFGF is itself an immediate early response to activin, a molecule that mimics the endogenous mesoderm-inducing signal. A model is proposed for the regulation of XmyoD within the early mesoderm. It is suggested that a maternal factor such as VegT induces the expression of a TGFß family member(s), which acts as the endogenous mesoderm inducing factor; this is likely a nodal related factor (Xnr1 and/or Xnr2). This endogenous mesoderm inducing factor is mimicked by activin, which induces the expression of eFGF directly. eFGF protein directly induces the expression of XmyoD, possibly acting through inhibition of a repressor. XmyoD is crucial in the specification of the myogenic cell lineage (Fisher, 2002).

In chick embryos, most if not all, replicating myoblasts present within the skeletal muscle masses express high levels of the FGF receptor FREK/FGFR4, suggesting an important role for this molecule during myogenesis. FGFR4 function during myogenesis was examined, and it has been demonstrated that inhibition of FGFR4, but not FGFR1 signaling, leads to a dramatic loss of limb muscles. All muscle markers analyzed (such as Myf5, MyoD and the embryonic myosin heavy chain) are affected. Inhibition of FGFR4 signal results in an arrest of muscle progenitor differentiation, which can be rapidly reverted by the addition of exogenous FGF, rather than a modification in their proliferative capacities. Conversely, over-expression of FGF8 in somites promotes FGFR4 expression and muscle differentiation in this tissue. Together, these results demonstrate that in vivo, myogenic differentiation is positively controlled by FGF signaling, a notion that contrasts with the general view that FGF promotes myoblast proliferation and represses myogenic differentiation. These data assign a novel role to FGF8 during chick myogenesis and demonstrate that FGFR4 signaling is a crucial step in the cascade of molecular events leading to terminal muscle differentiation (Maurics, 2002).

Skeletal muscle development involves an initial period of myoblast replication followed by a phase in which some myoblasts continue to proliferate while others undergo terminal differentiation. The latter process involves the permanent cessation of DNA synthesis, activation of muscle-specific gene expression, and fusion of single cells to generate multinucleated muscle fibers. The in vivo signals regulating the progression through all these steps remain unknown. Fibroblast growth factors (Fgfs) and Fgf receptors comprise a large family whose members have been shown to play multiple roles in the development of skeletal muscle in vitro. Exogenously applied Fgfs are able to stimulate proliferation and suppress myogenic differentiation in cell culture. Efforts have been made to determine the role played by Fgf-4 during limb myogenesis in vivo. Fgf-4 transcripts are located at both extremities of myotubes whereas the mRNAs of one of the Fgf receptors, Frek, are detected in mononucleated proliferating myoblasts surrounding the multinucleated fibers. Overexpression of mouse Fgf-4 (mFgf-4) using a replication-competent retrovirus, RCAS, leads to a down-regulation of muscle markers followed by an inhibition of terminal differentiation in limb muscles. Using quail/chick transplantations the muscle cells could be followed and a dramatic decrease in their number was found after exposure to mFgf-4. Interestingly ectopic mFgf-4 down-regulates Frek transcripts in limb muscle areas. It is concluded that overexpression of mFgf-4 inhibits myoblast proliferation, probably by down-regulating Frek mRNAs. This suggests a role for Fgf-4, located at the extremities of the myotubes, where it could be responsible for the absence of Frek mRNA in the muscle fibre (Edom-Vovard, 2001).

In vertebrates, tendons connect muscles to skeletal elements. Surgical experiments in the chick have underlined developmental interactions between tendons and muscles. Initial formation of tendons occurs autonomously with respect to muscle. However, further tendon development requires the presence of muscle. The molecular signals involved in these interactions remain unknown. In the chick limb, Fgf4 transcripts are located at the extremities of muscles, where the future tendons will attach. The putative role of muscle-Fgf4 on tendon development has been analyzed. Three general tendon markers, scleraxis (a bHLH transcription factor), tenascin, and Fgf8 were used to analyse the regulation of these tendon-associated molecules by Fgf4 under different experimental conditions. In the absence of Fgf4, in muscleless and aneural limbs, the expression of the three tendon-associated molecules, scleraxis, tenascin, and Fgf8, is down-regulated. Exogenous implantation of Fgf4 in normal, aneural, and muscleless limbs induces scleraxis and tenascin expression but not that of Fgf8. These results indicate that Fgf4 expressed in muscle is required for the maintenance of scleraxis and tenascin but not Fgf8 expression in tendons (Edom-Vovard, 2002).

Fgf-8 encodes a secreted signaling molecule mediating key roles in embryonic patterning. This study analyzes the expression pattern, regulation, and function of this growth factor in the paraxial mesoderm of the avian embryo. In the mature somite, expression of Fgf-8 is restricted to a subpopulation of myotome cells, comprising most, but not all, epaxial and hypaxial muscle precursors. Following ablation of the notochord and floor plate, Fgf-8 expression is not activated in the somites, in either the epaxial or the hypaxial domain, while ablation of the dorsal neural tube does not affect Fgf-8 expression in paraxial mesoderm. Contrary to the view that hypaxial muscle precursors are independent of regulatory influences from axial structures, these findings provide the first evidence for a regulatory influence of ventral, but not dorsal axial structures on the hypaxial muscle domain. Sonic hedgehog can substitute for the ventral neural tube and notochord in the initiation of Fgf-8 expression in the myotome. Fgf-8 protein leads to an increase in sclerotomal cell proliferation and enhances rib cartilage development in mature somites, whereas inhibition of Fgf signaling by SU 5402 causes deletions in developing ribs. These observations demonstrate: (1) a regulatory influence of the ventral axial organs on the hypaxial muscle compartment; (2) regulation of epaxial and hypaxial expression of Fgf-8 by Sonic hedgehog; and (3) independent regulation of Fgf-8 and MyoD in the hypaxial myotome by ventral axial organs. It is postulated that the notochord and ventral neural tube influence hypaxial expression of Fgf-8 in the myotome and that, in turn, Fgf-8 has a functional role in rib formation (Huang, 2003).

FGF, hematopoiesis, angiogenesis and endothelial cells

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor, synthesized and secreted by many differentiated cells in response to various stimuli, including hypoxia and growth factor exposure. Alternative splicing of vascular endothelial growth factor mRNA results in three distinct molecular forms: V189 and V165 or V121 which lack the exons 6 or 6 and 7, respectively. To clarify the functions of the 24-amino acid insertion, the biological activity of V165 was compared with that exerted by purified recombinant V189 and a synthetic peptide designed on the sequence encoded by exon 6 (Ex6P). V189 and Ex6P, but not V165, induce cell proliferation on corneal endothelial cells cultured in vitro. These effects are due to the release of fibroblast growth factor 2 (FGF2) stored in the extracellular matrix but not to direct interactions with FGF receptors, since V189 is inefficient on heparan sulfate-deficient cells constitutively expressing FGF-R1. Corneas incubated ex vivo with Ex6P solubilize 10-fold more FGF2 than a isocationic peptide containing a scrambled sequence. Ex6P elicits an angiogenic response in a corneal pocket assay, which is totally inhibited by addition of anti-FGF2 IgG. The angiogenic response to V189, but not to V165, is inhibited by FGF2 immunoneutralization. These findings demonstrate that the presence of the exon 6-encoded sequence confers VEGF with the ability to exert its biological effects through FGF2 signaling pathways (Jonca, 1997).

Cultures of Xenopus blastula animal caps were used to explore the hematopoietic effects of three candidate inducers of mesoderm: basic fibroblast growth factor (bFGF), bone morphogenetic proteins (BMPs) and activin A. In response to either bFGF or activin A, explants expand into egg-shaped structures: beneath an outer layer of epidermis, a ventral mesodermal lining surrounds a fluid-filled cavity containing 'blood-like cells'. Immunocytochemistry identifies some of these cells as early leukocytes, but erythrocytes are rare. BMP-2 or BMP-4 induces primitive erythrocytes as well as leukocytes, and a high concentration is required for these cells to differentiate in only a small proportion of explants. BMP-2 but not BMP-4 induces ventral mesoderm concomitantly. High concentrations of activin A dorsalizes explants, which contain infrequent leukocytes, and an optimal combination of activin A and bFGF causes differentiation of muscle with few blood cells. By contrast, BMP-2 or BMP-4 plus activin A synergistically increases the numbers of both leukocytes and erythrocytes. Explants treated with BMPs plus activin contain a well organized cell mass in which yolk-rich cells mix with blood cells and pigmented cells do not. BMP-2 plus bFGF also induces numerous leukocytes and fewer erythrocytes, but BMP-4 antagonizes the leukopoietic effect of bFGF. The data suggest that the signaling pathways these three factors use to induce leukopoiesis overlap and that erythropoiesis may be activated when inducers are present in combination (Miyanaga, 1999).

In adult vertebrates, fibroblast growth factor (FGF) synergizes with many hematopoietic cytokines to stimulate the proliferation of hematopoietic progenitors. In vertebrate development, the FGF signaling pathway is important in the formation of some derivatives of ventroposterior mesoderm. However, the function of FGF in the specification of the embryonic erythropoietic lineage has remained unclear. The role of FGF in the specification of the erythropoietic lineage in the Xenopus embryo is addressed in this paper. Ventral injection of embryonic FGF (eFGF) mRNA at as little as 10 pg at the four-cell stage suppresses ventral blood island (VBI) formation, whereas expression of the dominant negative form of the FGF receptor in the lateral mesoderm, where physiologically no blood tissue is formed, results in a dramatic expansion of the VBI. Similar results were observed in isolated ventral marginal zones and animal caps. Bone morphogenetic protein-4 (BMP-4) is known to induce erythropoiesis in the Xenopus embryo. Therefore, an examination was carried out of how the BMP-4 and FGF signaling pathways might interact in the decision of ventral mesoderm to form blood. eFGF inhibits BMP-4-induced erythropoiesis by differentially regulating expression of the BMP-4 downstream effectors GATA-2 and PV.1. GATA-2, which stimulates erythropoiesis, is suppressed by FGF. PV.1, which inhibits blood development, is enhanced by FGF. Additionally, PV.1 and GATA-2 negatively regulate transcription of one another. Thus, BMP-4 induces two transcription factors that have opposing effects on blood development. The FGF and BMP-4 signaling pathways interact to regulate the specification of the erythropoietic lineage (Xu, 1999).

Fibroblast growth factor-1 (FGF-1), a prototype member of the heparin-binding growth factor family, is a potent mitogen for vascular endothelial cells and a variety of other cell types. FGF-1 can induce the expression of the platelet-derived growth factor-A chain (PDGF-A) gene in endothelial cells; however, the underlying transcriptional mechanisms are not known. A 16-bp element, located 55 to 71 bp upstream of the transcriptional start site, is required for FGF-1-inducible promoter-dependent expression of PDGF-A. This region contains nucleotide recognition elements for the early growth response gene product, early growth response factor-1 (Egr-1), and the related zinc-finger transcription factor, Sp1. FGF-1 induces Egr-1 mRNA expression within 30 minutes. Egr-1 protein accumulates in the nuclei of endothelial cells exposed to the growth factor, whereas levels of Sp1 do not change. Egr-1 binds to the FGF-1 response element in the proximal PDGF-A promoter in a specific and time-dependent manner. These findings indicate that Egr-1 plays a key regulatory role in FGF-1-inducible endothelial PDGF-A expression and implicate this transcription factor in pathological settings in which these mitogens are both expressed (Delbridge, 1997).

BLast Colony Forming Cells (BL-CFCs) from in vitro differentiated embryonic stem (ES) cells represent the common progenitor of hematopoietic and endothelial cells, the hemangioblast. Access to this initial cell population committed to the hematopoietic lineage provides a unique opportunity to characterize hematopoietic commitment events. Here, BL-CFC expresses the receptor tyrosine kinase, Flk1 (the VEGF receptor), and thus advantage was taken of the BL-CFC assay, as well as fluorescent activated cell sorter (FACS) analysis for Flk1 + cells to determine quantitatively if mesoderm-inducing factors promote hematopoietic lineage development. Moreover, ES lines carrying targeted mutations for fibroblast growth factor receptor-1 (fgfr1), a receptor for basic fibroblast growth factor (bFGF), as well as scl, a transcription factor, were analyzed for their potential to generate BL-CFCs and Flk1 + cells, to further define events leading to hemangioblast development. bFGF together with activin A appear to have an additive or synergistic effect on BL-CFC generation; activin A-mediated BL-CFC generation requires a Fgfr1 signal since the administration of activin A does not rescue the BL-CFC development in fgfr1-/- embryoid bodies [in vitro differentiated progeny (embryoid bodies, EBs) of embryonic stem (ES) cells]. These results are consistent with the findings in Xenopus that activin A and bFGF synergize in mesoderm induction and that activin A requires a bFGF-mediated signal. The data suggest that bFGF-mediated signaling is critical for the proliferation of the hemangioblast and that cells expressing both Flk1 and SCL may represent the hemangioblast (Faloon, 2000).

FGF and bone growth

Fibroblast growth factor receptor 3 (FGFR3) is a key regulator of skeletal growth. Activating mutations in Fgfr3cause achondroplasia, the most common genetic form of dwarfism in humans. Little is known about the mechanism by which FGFR3 inhibits bone growth and how FGFR3 signaling interacts with other signaling pathways that regulate endochondral ossification. To understand these mechanisms, the expression of an activated FGFR3 was targeted to growth plate cartilage in mice using regulatory elements from the collagen II gene. As with humans carrying the achondroplasia mutation, the resulting transgenic mice are dwarfed, with axial, appendicular and craniofacial skeletal hypoplasia. FGFR3 inhibits endochondral bone growth by markedly inhibiting chondrocyte proliferation and by slowing chondrocyte differentiation. Significantly, FGFR3 downregulates the Indian hedgehog (Ihh) signaling pathway and Bmp4 expression in both growth plate chondrocytes and in the perichondrium. Conversely, Bmp4 expression is upregulated in the perichondrium of Fgfr3-/- mice. These data support a model in which Fgfr3 is an upstream negative regulator of the Hedgehog (Hh) signaling pathway. Additionally, Fgfr3 may coordinate the growth and differentiation of chondrocytes with the growth and differentiation of osteoprogenitor cells by simultaneously modulating Bmp4 and patched expression in both growth plate cartilage and in the perichondrium (Naski, 1998).

Mutations in the Fgfr1-Fgfr3 and Twist genes are known to cause craniosynostosis (premature fusion of the cranial sutures leading to skull deformity), the former by constitutive activation and the latter by haploinsufficiency. Although clinically achieving the same end result, the premature fusion of the calvarial (skull) bones, it is not known whether these genes lie in the same or independent pathways during calvarial bone development and later in suture closure. Fgfr2c is expressed at the osteogenic fronts of the developing calvarial bones. When FGF is applied via beads to the osteogenic fronts, suture closure is accelerated. In order to investigate the role of FGF signaling during mouse calvarial bone and suture development, detailed expression analysis of the splicing variants of Fgfr1-Fgfr3 and Fgfr4, as well as their potential ligand Fgf2 were performed. The IIIc splice variants of Fgfr1-Fgfr3 as well as the IIIb variant of Fgfr2 are expressed by differentiating osteoblasts at the osteogenic fronts (E15). In comparison to Fgf9, Fgf2 shows a more restricted expression pattern, being primarily expressed in the sutural mesenchyme between the osteogenic fronts. A detailed expression analysis has been carried out of the helix-loop-helix factors (HLH) Twist and Id1 during calvaria and suture development (E10-P6). Twist and Id1 are expressed by early preosteoblasts, in patterns that overlap those of the FGF ligands, but as these cells differentiate their expression dramatically decreases. signaling pathways were further studied in vitro, in E15 mouse calvarial explants. Beads soaked in FGF2 induce Twist and inhibit Bsp, a marker of functioning osteoblasts. Meanwhile, BMP2 upregulates Id1. Id1 is a dominant negative HLH protein thought to inhibit basic HLH proteins such as Twist. In Twist+/- mice, Fgfr2 protein expression is altered (Rice, 2000).

It is proposed that FGFs have functions at several stages of osteoblast differentiation. FGF2 has both inhibitory and stimulatory effects on osteoblast activity and evidence is presented that the inhibitory effects may be via a Twist regulated pathway. In line with Twist having a negative regulatory effect on osteoblast differentiation, the Twist mutation causing craniosynostosis is thought to be a loss-of-function mutation. Thus, Twist would appear to be upstream of FGFR/FGF signaling, though whether it is inhibitory or stimulatory cannot yet be definitively concluded. FGF may also act at a later stage in osteoblast differentiation, with both excess FGF and overactivation of FGF receptors causing an acceleration of suture closure. It is known that Id inhibits bHLH factors such as Twist, and that BMP2 induces osteoblast maturation. BMP2 is shown to stimulate Id and it is therefore postulated that the effects of BMP2 on osteoblast differentiation may be via Id's inhibition of Twist, thereby promoting cell differentiation instead of proliferation. However, it is known that overexpression of Id decreases the activity of the osteocalcin promoter, and that BMP can cause an increase in calvarial mesenchymal tissue volume. BMP2 and Id may therefore also act independently, stimulating osteoblast proliferation (Rice, 2000).

Gain of function mutations in fibroblast growth factor (FGF) receptors cause chondrodysplasia and craniosynostosis syndromes. Identification of the ligands interacting with FGF receptors (FGFRs) in developing bone has remained elusive, and the mechanisms by which FGF signaling regulates endochondral, periosteal, and intramembranous bone growth are as yet not known. This study shows that Fgf18 is expressed in the perichondrium and that mice homozygous for a targeted disruption of Fgf18 exhibit a growth plate phenotype similar to that observed in mice lacking Fgfr3 and an ossification defect at sites that express Fgfr2. Mice lacking either Fgf18 or Fgfr3 exhibit expanded zones of proliferating and hypertrophic chondrocytes and increased chondrocyte proliferation, differentiation, and Indian hedgehog signaling. These data suggest that FGF18 acts as a physiological ligand for FGFR3. In addition, mice lacking Fgf18 display delayed ossification and decreased expression of osteogenic markers, phenotypes not seen in mice lacking Fgfr3. These data demonstrate that FGF18 signals through another FGFR to regulate osteoblast growth. Signaling to multiple FGFRs positions FGF18 to coordinate chondrogenesis in the growth plate with osteogenesis in cortical and trabecular bone (Liu, 2002).

Fibroblast growth factor (FGF) signaling is involved in skeletal development of the vertebrate. Gain-of-function mutations of FGF receptors (FGFR) cause craniosynostosis (premature fusion of the skull) and dwarfism syndromes. Disruption of Fgfr3 results in prolonged growth of long bones and vertebrae. Fgf18 is expressed in and required for osteogenesis and chondrogenesis in the mouse embryo. Fgf18 is expressed in both osteogenic mesenchymal cells and differentiating osteoblasts during calvarial bone development. In addition, Fgf18 is expressed in the perichondrium and joints of developing long bones. In calvarial bone development of Fgf18-deficient mice generated by gene targeting, the progress of suture closure is delayed. Furthermore, proliferation of calvarial osteogenic mesenchymal cells is decreased, and terminal differentiation to calvarial osteoblasts is specifically delayed. Delay of osteogenic differentiation is also observed in the developing long bones of this mutant. Conversely, chondrocyte proliferation and the number of differentiated chondrocytes are increased. Therefore, FGF18 appears to regulate cell proliferation and differentiation positively in osteogenesis and negatively in chondrogenesis (Ohbayashi, 2002).

The formation of cartilage elements in the developing vertebrate limb, where they serve as primordia for the appendicular skeleton, is preceded by the appearance of discrete cellular condensations. Control of the size and spacing of these condensations is a key aspect of skeletal pattern formation. Limb bud cell cultures grown in the absence of ectoderm form continuous sheet-like masses of cartilage. With the inclusion of ectoderm, these cultures produce one or more cartilage nodules surrounded by zones of noncartilaginous mesenchyme. Ectodermal fibroblast growth factors (FGF2 and FGF8), but not a mesodermal FGF (FGF7), substitute for ectoderm in inhibiting chondrogenic gene expression, with some combinations of the two ectodermal factors leading to well-spaced cartilage nodules of relatively uniform size. Treatment of cultures with SU5402, an inhibitor FGF receptor tyrosine kinase activity, renders FGFs ineffective in inducing perinodular inhibition. Inhibition of production of FGF receptor 2 (FGFR2) by transfection of wing and leg cell cultures with antisense oligodeoxynucleotides, blocks appearance of ectoderm- or FGF-induced zones of perinodular inhibition of chondrogenesis, and, when introduced into the limb buds of developing embryos, this leads to shorter, thicker, and fused cartilage elements. Because FGFR2 is expressed mainly at sites of precartilage condensation during limb development in vivo and in vitro, these results suggest that activation of FGFR2 by FGFs during development elicits a lateral inhibitor of chondrogenesis that limits the expansion of developing skeletal elements (Moftah, 2002).

FGF and heart development

Previous studies have identified two signaling interactions regulating cardiac myogenesis in avians, a hypoblast-derived signal acting on epiblast and mediated by activin or a related molecule and an endoderm-derived signal acting on mesoderm and involving BMP-2. In this study, experiments were designed to investigate the temporal relationship between these signaling events and the potential role of other TGFbeta superfamily members in regulating early steps of heart muscle development. While activin or TGFbeta can potently induce cardiac myogenesis in pregastrula epiblast, they show no capacity to convert noncardiogenic mesoderm toward a myocardial phenotype. Conversely, BMP-2 or BMP-4, in combination with FGF-4, can readily induce cardiac myocyte formation in posterior mesoderm, but shows no capacity to induce cardiac myogenesis in epiblast cells. Activin/TGFbeta and BMP-2/BMP-4 therefore have distinct and reciprocal cardiac-inducing capacities that mimic the tissues in which they are expressed, the pregastrula hypoblast and anterior lateral endoderm, respectively. Experiments with noggin and follistatin provide additional evidence indicating that BMP signaling lies downstream of an activin/TGFbeta signal in the cardiac myogenesis pathway. In contrast to the cardiogenic-inducing capacities of BMP-2/BMP-4 in mesoderm, however, BMP-2 or BMP-4 inhibits cardiac myogenesis prior to stage 3, demonstrating multiple roles for BMPs in mesoderm induction. These and other published studies suggest a signaling cascade in which a hypoblast-derived activin/TGFbeta signal is required prior to and during early stages of gastrulation, regulated both spatially and temporally by an interplay between BMPs and their antagonists. Later cardiogenic signals arising from endoderm, and perhaps transiently from ectoderm, and mediated in part by BMPs, act on emerging mesoderm within cardiogenic regions to activate or enhance expression of cardiogenic genes such as GATA and cNkx family members, leading to cardiac myocyte differentiation (Ladd, 1998).

Vertebrate heart development is initiated from bilateral lateral plate mesoderm that expresses the Nkx2.5 and GATA4 transcription factors, but the extracellular signals specifying heart precursor gene expression are not known. The secreted signaling factor Fgf8 is expressed in and required for development of the zebrafish heart precursors, particularly during initiation of cardiac gene expression. fgf8 is mutated in acerebellar (ace) mutants, and homozygous mutant embryos do not establish normal circulation, although vessel formation is only mildly affected. In contrast, heart development, in particular of the ventricle, is severely abnormal in acerebellar mutants. Several findings argue that Fgf8 has a direct function in development of cardiac precursor cells: fgf8 is expressed in cardiac precursors and later in the heart ventricle. Fgf8 is required for the earliest stages of nkx2.5 and gata4 expression in cardiac precursors, but not for gata6. Cardiac gene expression is restored in acerebellar mutant embryos by injecting fgf8 mRNA, or by implanting a Fgf8-coated bead into the heart primordium. Pharmacological inhibition of Fgf signaling during formation of the heart primordium phenocopies the acerebellar heart phenotype, confirming that Fgf signaling is required independent of earlier functions during gastrulation. These findings show that fgf8/acerebellar is required for induction and patterning of myocardial precursors (Reifers, 2000).

The avian heart develops from paired primordia located in the anterior lateral mesoderm of the early embryo. Previous studies have found that the endoderm adjacent to the cardiac primordia plays an important role in heart specification. The current study provides evidence that fibroblast growth factor (Fgf) signaling contributes to the heart-inducing properties of the endoderm. Fgf8 is expressed in the endoderm adjacent to the precardiac mesoderm. Removal of endoderm results in a rapid downregulation of a subset of cardiac markers, including Nkx2.5 and Mef2c. Expression of these markers can be rescued by supplying exogenous Fgf8. In addition, application of ectopic Fgf8 results in ectopic expression of cardiac markers. Expression of cardiac markers is expanded only in regions where bone morphogenetic protein (Bmp) signaling is also present, suggesting that cardiogenesis occurs in regions exposed to both Fgf and Bmp signaling. Finally, evidence is presented that Fgf8 expression is regulated by particular levels of Bmp signaling. Application of low concentrations of Bmp2 results in ectopic expression of Fgf8, while application of higher concentrations of Bmp2 result in repression of Fgf8 expression. Together, these data indicate that Fgf signaling cooperates with Bmp signaling to regulate early cardiogenesis (Alsan, 2002).

Fibroblast growth factor 8 (Fgf8) is expressed in many domains of the developing embryo. Globally decreased FGF8 signaling during murine embryogenesis results in a hypomorphic phenotype with a constellation of heart, outflow tract, great vessel and pharyngeal gland defects that phenocopies human deletion 22q11 syndromes, such as DiGeorge. It is postulated that these Fgf8 hypomorphic phenotypes result from disruption of local FGF8 signaling from pharyngeal arch epithelia to mesenchymal cells populating and migrating through the third and fourth pharyngeal arches. To test this hypothesis, and to determine whether the pharyngeal ectoderm and endoderm Fgf8 expression domains have discrete functional roles, conditional mutagenesis of Fgf8 was performed using novel Crerecombinase drivers to achieve domain-specific ablation of Fgf8 gene function in the pharyngeal arch ectoderm and endoderm. Remarkably, ablating FGF8 protein in the pharyngeal arch ectoderm causes failure of formation of the fourth pharyngeal arch artery that results in aortic arch and subclavian artery anomalies in 95% of mutants; these defects recapitulate the spectrum and frequency of vascular defects reported in Fgf8 hypomorphs. Surprisingly, no cardiac, outflow tract or glandular defects were found in ectodermal-domain mutants, indicating that ectodermally derived FGF8 has essential roles during pharyngeal arch vascular development distinct from those in cardiac, outflow tract and pharyngeal gland morphogenesis. By contrast, ablation of FGF8 in the third and fourth pharyngeal endoderm and ectoderm causes glandular defects and bicuspid aortic valve, which indicates that the FGF8 endodermal domain has discrete roles in pharyngeal and valvar development. These results support the hypotheses that local FGF8 signaling from the pharyngeal epithelia is required for pharyngeal vascular and glandular development, and that the pharyngeal ectodermal and endodermal domains of FGF8 have separate functions (Macatee, 2003).

Dysmorphogenesis of the cardiac outflow tract (OFT) causes many congenital heart defects, including those associated with DiGeorge syndrome. Genetic manipulation in the mouse and mutational analysis in patients have shown that Tbx1, a T-box transcription factor, has a key role in the pathogenesis of this syndrome. Tbx1 function during OFT development have been dissected using genetically modified mice and tissue-specific deletion, and have defined a dual role for this protein in OFT morphogenesis. Tbx1 regulates cell contribution to the OFT by supporting cell proliferation in the secondary heart field, a source of cells fated to the OFT. This process might be regulated in part by Fgf10, which is a direct target of Tbx1 in vitro. Tbx1 expression is required in cells expressing Nkx2.5 for the formation of the aorto-pulmonary septum, which divides the aorta from the main pulmonary artery. These results explain why aortic arch patterning defects and OFT defects can occur independently in individuals with DiGeorge syndrome. Furthermore, the data link the function of the secondary heart field to congenital heart disease (Xu, 2004).

Table of contents

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

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