FGF receptor 1


Table of contents

FGF receptors and limb specification and development

Fgf-10-deficient mice (Fgf-10(-/-)) were generated to determine the role(s) of Fgf-10 in vertebrate development. Limb bud initiation was abolished in Fgf-10(-/-) mice. Strikingly, Fgf-10-/- fetuses continue to develop until birth, despite the complete absence of both forelimbs and hindlimbs. Fgf-10 is necessary for apical ectodermal ridge (AER) formation and acts epistatically upstream of Fgf-8, the earliest known AER marker in mice. Fgf-10-/- mice exhibit perinatal lethality associated with complete absence of lungs. Although tracheal development is normal, main-stem bronchial formation, as well as all subsequent pulmonary branching morphogenesis, is completely disrupted. Lack of lungs and main-stem bronchi in Fgf-10-/- mice is very reminiscent of the Drosophila mutant bnl. Both genetic and biochemical evidence indicates that Bnl is a ligand for Breathless (Btl), an Fgf receptor. Drosophila btl mutants exhibited a phenotype that was very similar to that of bnl. Mammalian Fgfr2b is highly expressed in the epithelium throughout embryonic lung development. Transgenic mice expressing a dominant-negative form of FGFR2b splice variant under control of the SP-C promotor exhibit perinatal lethality and failed to develop lungs, indicating that FGFR2b is a receptor necessary for pulmonary branching morphogenesis. Ligands of this receptor include FGF-1, FGF-7, and FGF-10, and all three have been shown to promote expansion and/or budding of endodermal cells in lung explant studies. Fgfr2b transgenic mice exhibit trachea formation and bifurcation of main-stem bronchi, unlike the Fgf-10 knockout mice, which only develop a trachea without further branching. This difference was most likely caused by spatial and temporal differences between Fgf-10 expression and SP-C promotor activity. The SP-C promotor drives transgene expression in distal lung epithelium starting at E10. In contrast, Fgf-10 is already expressed in the distal mesenchymal cells of developing respiratory tract buds at E9.5. Presumably, by E10-E10.5, the formation of the primordial bronchi has already occurred. The impaired pulmonary development observed in Fgfr2b transgenic mice, coupled with similarities in pulmonary phenotypes of Fgf-10 knockout mice and Drosophila bnl and btl mutants, suggests striking functional similarities in the signaling pathways of mammalian Fgf-10 and Drosophila bnl (Min, 1998).

The Fgf-8 gene is expressed in developing limb and craniofacial structures, regions known to be important for growth and patterning of the mouse embryo. Although Fgf-8 is alternatively spliced to generate at least 7 secreted isoforms that differ only at their mature amino terminus, the biological significance of these multiple isoforms is not known. Multiple FGF-8 isoforms are present at sites of Fgf-8 expression during mouse development. To address the possibility that the FGF-8 isoforms might interact with different fibroblast growth factor receptors, recombinant FGF-8 protein isoforms were prepared. Recombinant FGF-8b and FGF-8c activate the 'c' splice form of FGFR3, and FGFR4, while FGF-8b also efficiently activates the 'c' splice form of FGFR2. No activity is detected for recombinant or cell expressed FGF-8a. Furthermore, none of the isoforms tested interact efficiently with 'b' splice forms of FGFR1-3, or the 'c' splice form of FGFR1. These results indicate that the FGF-8b and FGF-8c isoforms, produced by ectodermally derived epithelial cells, interact with mesenchymally expressed fibroblast growth factor receptors. FGF-8b and FGF-8c may therefore provide a mitogenic signal to the underlying mesenchyme during limb and craniofacial development (MacArthur, 1995).

Pattern in the developing limb depends on signaling by polarizing region mesenchyme cells, which are located at the posterior margin of the bud tip. In the intact bud, connexin 43 (Cx43) and Cx32 gap junctions are found in higher densities between distal posterior mesenchyme cells at the tip of the bud than between either distal anterior or proximal mesenchyme cells. These gradients disappear when the apical ectodermal ridge (AER) is removed. Fibroblast growth factor 4 (FGF4) produced by posterior AER cells controls signaling by polarizing cells. FGF4 doubles gap junction density and substantially improves functional coupling between cultured posterior mesenchyme cells. FGF4 has no effect on cultured anterior mesenchyme, suggesting that any effects of FGF4 on responding anterior mesenchyme cells are not mediated by a change in gap junction density or functional communication through gap junctions. In condensing mesenchyme cells, connexin expression is not affected by FGF4. Posterior mesenchyme cells maintained in FGF4 under conditions that increase functional coupling also maintain polarizing activity at in vivo levels. Without FGF4, polarizing activity is reduced and the signaling mechanism changes. It is concluded that FGF4 regulation of cell-cell communication and polarizing signaling are intimately connected (Makarenkova, 1997).

FGFR2 is a membrane-spanning tyrosine kinase that serves as a high affinity receptor for several members of the fibroblast growth factor (FGF) family. To explore functions of FGF/FGFR2 signals in development, FGFR2 has been mutated by deleting the entire immunoglobin-like domain III of the receptor. Murine FGFR2 is essential for chorioallantoic fusion and placenta trophoblast cell proliferation. Fgfr2 mutant embryos display two distinct defects that result in failure to form a functional placenta. About one third of the mutants fail to form the chorioallantoic fusion junction and the remaining mutants do not have the labyrinthine portion of the placenta. Consequently, all mutants die at 10-11 days of gestation. Interestingly, mutant embryos do not form limb buds. Consistent with this defect, the expression of Fgf8, an apical ectodermal factor, is absent in the mutant presumptive limb ectoderm, and the expression of Fgf10, a mesenchymally expressed limb bud initiator, is down regulated in the underlying mesoderm. These findings provide direct genetic evidence that FGF/FGFR2 signals are absolutely required for vertebrate limb induction and that an FGFR2 signal is essential for the reciprocal regulation loop between FGF8 and FGF10 during limb induction (Xu, 1998).

Following amputation of a urodele limb or teleost fin, the formation of a blastema is a crucial step in facilitating subsequent regeneration. Early caudal fin regenerative events can be separated into four stages. (1) During the first 12 h, epidermal cells migrate to cover the stump. (2) Within the next 12 h, the wound epidermis thickens, while fibroblasts and scleroblasts located within one or two bone segments proximal to the amputation plane lose their dense organization and show signs of distal migration. (3) Next, these mesenchymal cells organize and proliferate to form a blastema, a mass of undifferentiated tissue, just distal to the ray stumps. (4) During the outgrowth stage, proximal cells of the regeneration blastema differentiate to participate in bone deposition, while distal cells divide and maintain outgrowth. Using the zebrafish caudal fin regeneration model, the hypothesis that fibroblast growth factors initiate blastema formation from fin mesenchyme was examined. fibroblast growth factor receptor 1 (fgfr1) is expressed in mesenchymal cells underlying the wound epidermis during blastema formation and in distal blastemal tissue during regenerative outgrowth. fgfr1 transcripts colocalize with those of msxb and msxc, putative markers for undifferentiated, proliferating cells. A zebrafish Fgf member, designated wfgf, is expressed in the regeneration epidermis during outgrowth. Furthermore, a specific inhibitor of Fgfr1, applied immediately following fin amputation, blocks blastema formation without obvious effects on wound healing. This inhibitor blocks the proliferation of blastemal cells and the onset of msx gene transcription. Inhibition of Fgf signaling during ongoing fin regeneration prevents further outgrowth while downregulating the established expression of blastemal msx genes and epidermal sonic hedgehog. These findings indicate that zebrafish fin blastema formation and regenerative outgrowth require Fgf signaling (Poss, 2000).

It is proposed that following amputation and wound healing, mesenchymal cells disorganize and begin to migrate toward the amputation plane. At the epidermal-mesenchymal junction, Fgf molecules synthesized in the wound epidermis bind to mesenchymal Fgfr1. Signaling by Fgfr1 triggers proliferation and the induction or maintenance of msxb and msxc expression in these cells, and a blastema forms. During later stages, Wfgf and/or other Fgfs are released from the distalmost epidermal cells and signal through blastemal Fgfr1 to maintain msxb/c expression and cell division, which promotes outgrowth. Meanwhile, Fgfs activate Fgfrs in basal layer epidermal cells to maintain shh transcription during outgrowth. Shh released from these cells is thought to help direct new bone deposition by scleroblasts (Poss, 2000).

Mice deficient for FgfR2-IIIb were generated by placing translational stop codons and an IRES-LacZ cassette into exon IIIb of FgfR2. Expression of the alternatively spliced receptor isoform, FgfR2-IIIc, is not affected in mice deficient for the IIIb isoform. FgfR2-IIIb -/-lacZ mice survive to term but show dysgenesis of the kidneys, salivary glands, adrenal glands, thymus, pancreas, skin, otic vesicles, glandular stomach, and hair follicles, and agenesis of the lungs, anterior pituitary, thyroid, teeth, and limbs. Detailed analysis of limb development revealed an essential role for FgfR2-IIIb in maintaining the AER. Its absence does not prevent expression of Fgf8, Fgf10, Bmp4, and Msx1, but does prevent induction of Shh and Fgf4, indicating that these genes are downstream targets of FgfR2-IIIb activation. In the absence of FgfR2-IIIb, extensive apoptosis of the limb bud ectoderm and mesenchyme occurs between E10 and E10.5, providing evidence that Fgfs act primarily as survival factors. It is proposed that FgfR2-IIIb is not required for limb bud initiation, but is essential for its maintenance and growth (Revest, 2001).

Mouse Twist is essential for cranial neural tube, limb and somite development. To identify the molecular defects disrupting limb morphogenesis, expression of mesenchymal transcription factors involved in patterning and the cell-cell signaling cascades controlling limb bud development were examined. These studies establish that Twist is essential for maintenance and progression of limb bud morphogenesis. In particular, the SHH/FGF signaling feedback loop operating between the polarizing region and the apical ectodermal ridge (AER) is disrupted. These defects in epithelial-mesenchymal signaling are most likely a direct consequence of disrupted fibroblast growth factor (FGF) signaling in Twist-deficient limb buds. In early limb buds, down-regulation of Fgf receptor 1 and Fgf10 expression in the mesenchyme occurs concurrent with loss of Fgf4 and Fgf8 expression in the AER. Finally, Twist function, most likely by regulating FGF signaling, is required for cell survival since apoptotic cells are detected in posterior and distal limb bud mesenchyme (Zuniga, 2002).

FGF receptor and breast development

Separated normal human breast epithelial and myoepithelial cells were examined for the presence of basic fibroblast growth factor (FGF2) and its receptors, both low (heparan sulfate proteoglycans) and high affinity (FGFR1), and for the effects of FGF2 on the proliferation of both cell types. These cells differ markedly in their synthesis and response to FGF2. mRNA for FGF2 is found only in the myoepithelial cells, whereas FGF2 protein is present in both epithelial and myoepithelial cells. FGF2 has no effect on the proliferation of myoepithelial cells, but it maintains the survival of the separated epithelial cells in low serum and stimulates their growth in 5% and 10% FCS. Immunostainable FGFR1 is present in epithelial cells and, to a lesser extent, in myoepithelial cells. Low-affinity binding sites for FGF2 are synthesized by epithelial and myoepithelial cells, but myoepithelial cells possess a greater proportion of higher-affinity heparan sulfate proteoglycans. These results indicate that myoepithelial cell-derived FGF2 may be an important paracrine factor controlling epithelial cell survival and growth in the normal human breast (Gomm, 1997).

A mouse mammary tumor virus promoter was used to express two dominant negative (DN) fibroblast growth factor receptor (FGFR) isoforms in the mammary epithelium of transgenic mice. While expression of DN-FGFR1(IIIc) shows no discernible phenotype, a similar kinase negative form of FGFR2(IIIb) causes a marked impairment of lobuloalveolar development. The growth retardation is apparent by mid-pregnancy and persists in the post-partum glands. Despite the substantial under-development of the mammary gland, there is a measurable lactational response, but it is insufficient to properly sustain the new-born pups. These findings demonstrate that fibroblast growth factor signaling is necessary for pregnancy-dependent lobuloalveolar development of the mammary gland (Jackson, 1997).

During appendage regeneration in urodeles and teleosts, tissue replacement is precisely regulated such that only the appropriate structures are recovered, a phenomenon referred to as positional memory. It is believed that there exists, or is quickly established after amputation, a dynamic gradient of positional information along the proximodistal (PD) axis of the appendage that assigns region-specific instructions to injured tissue. These instructions specify the amount of tissue to regenerate, as well as the rate at which regenerative growth is to occur. A striking theme among many species is that the rate of regeneration is more rapid in proximally amputated appendages compared with distal amputations. However, the underlying molecular regulation is unclear. This study identifies position-dependent differences in the rate of growth during zebrafish caudal fin regeneration. These growth rates correlate with position-dependent differences in blastemal length, mitotic index and expression of the Fgf target genes mkp3, sef and spry4. To address whether PD differences in amounts of Fgf signaling are responsible for position-dependent blastemal function, transgenic fish were generated in which Fgf receptor activity can be experimentally manipulated. It was found that the level of Fgf signaling exhibits strict control over target gene expression, blastemal proliferation and regenerative growth rate. These results demonstrate that Fgf signaling defines position-dependent blastemal properties and growth rates for the regenerating zebrafish appendage (Lee, 2005).

Fibroblast growth factors (FGFs) and their receptors have been implicated in limb development. However, because of early post-implantation lethality associated with fibroblast growth factor receptor 1 (FGFR1) deficiency, the role of this receptor in limb development remains elusive. To overcome embryonic lethality, a conditional knockout of Fgfr1 was performed using the Cre-LoxP approach. Cre-mediated deletion of Fgfr1 in limb mesenchyme, beginning at a time point slightly after the first sign of initial budding, primarily affects formation of the first one or two digits. In contrast, deletion of Fgfr1 at an earlier stage, prior to thickening of limb mesenchyme, results in more severe defects, characterized by malformation of the AER, diminished Shh expression and the absence of the majority of the autopod skeletal elements. FGFR1 deficiency does not affect cell proliferation. Instead, it triggers cell death and leads to alterations in expression of a number of genes involved in apoptosis and digit patterning, including increased expression of Bmp4, Dkk1 and Alx4, and downregulation of MKP3. These data demonstrate that FGF/FGFR1 signals play indispensable roles in the early stages of limb initiation, eliciting a profound effect on the later stages of limb development, including cell survival, autopod formation and digit patterning (. Li, 2005 ).

Fibroblast growth factor receptor 1 (FGFR1) has been implicated in limb development, but the precise nature and complexity of its role has not been defined. This study dissects Fgfr1 function in mouse limb by conditional inactivation of Fgfr1 using two different Cre recombinase-expressing lines. Use of the T (brachyury)-cre line led to Fgfr1 inactivation in all limb bud mesenchyme (LBM) cells during limb initiation. This mutant reveals FGFR1 function in two phases of limb development. In a nascent limb bud, FGFR1 promotes the length of the proximodistal (PD) axis while restricting the dimensions of the other two axes. It also serves an unexpected role in limiting LBM cell number in this early phase. Later on during limb outgrowth, FGFR1 is essential for the expansion of skeletal precursor population by maintaining cell survival. Use of mice carrying the sonic hedgehogcre (Shhcre) allele led to Fgfr1 inactivation in posterior LBM cells. This mutant allows a test of the role of Fgfr1 in gene expression regulation without disturbing limb bud growth. These data show that during autopod patterning, FGFR1 influences digit number and identity, probably through cell-autonomous regulation of Shh expression. This study of these two Fgfr1 conditional mutants has elucidated the multiple roles of FGFR1 in limb bud establishment, growth and patterning (Verheyden, 2005).

Fgf receptor and lens induction

Fibroblast growth factor receptor (Fgfr) signaling plays a role in lens induction. Three distinct experimental strategies were used: (1) using small-molecule inhibitors of Fgfr kinase activity, it has been shown that both the transcription level and protein expression of Pax6, a transcription factor critical for lens development, is diminished in the presumptive lens ectoderm; (2) transgenic mice (designated Tfr7) that express a dominant-negative Fgf receptor exclusively in the presumptive lens ectoderm show defects in formation of the lens placode at E9.5 but in addition, showe reduced levels of expression for Pax6, Sox2 and Foxe3, all markers of lens induction; (3) by performing crosses between Tfr7 transgenic and Bmp7-null mice, it has been shown that there is a genetic interaction between Fgfr and Bmp7 signaling at the induction phases of lens development. This is manifested as exacerbated lens development defects and lower levels of Pax6 and Foxe3 expression in Tfr7/Tfr7, Bmp7+/- mice, when compared with Tfr7/Tfr7 mice alone. Since Bmp7 is an established lens induction signal, this provides further evidence that Fgfr activity is important for lens induction. This analysis establishes a role for Fgfr signaling in lens induction and defines a genetic pathway in which Fgfr and Bmp7 signaling converge on Pax6 expression in the lens placode with the Foxe3 and Sox2 genes lying downstream (Faber, 2001).

The epithelial b variant of Fgfr2 is active in the entire surface ectoderm of the early embryo, and later in the limb ectoderm and AER, where it is required for limb outgrowth. Since limb buds do not form in the absence of Fgfr2, chimera analysis was used to investigate the mechanism of action of this receptor in limb development. ES cells homozygous for a loss-of-function mutation of Fgfr2 that carry a ß-galactosidase reporter were aggregated with normal pre-implantation embryos. Chimeras with a high proportion of mutant cells do not form limbs, whereas those with a moderate proportion form limb buds with a lobular structure and a discontinuous AER. Where present, the AER do not contain mutant cells, although mutant cells localize to the adjacent surface ectoderm and limb mesenchyme. In the underlying mesenchyme of AER-free areas, cell proliferation is reduced, and transcription of Shh and Msx1 is diminished. En1 expression in the ventral ectoderm is discontinuous and exhibits ectopic dorsal localization, whereas Wnt7a expression is diminished in the dorsal ectoderm but remains confined to that site. En1 and Wnt7a are not expressed in non-chimeric Fgfr2-null mutant embryos, revealing that they are downstream of Fgfr2. In late gestation chimeras, defects presented in all three limb segments as bone duplications, bone loss or ectopic outgrowths. It is suggested that Fgfr2 is required for AER differentiation, as well as for En1 and Wnt7a expression. This receptor also mediates signals from the limb mesenchyme to the limb ectoderm throughout limb development, affecting the position and morphogenesis of precursor cells in the dorsal and ventral limb ectoderm, and AER (Gorivodsky, 2003).

FGF receptor and lens fiber cell differentiation

To determine if fibroblast growth factor signaling mechanisms are required for terminal differentiation and survival of lens fiber cells, the effects of expressing truncated fibroblast growth factor receptors (tFGFRs) in different regions of the developing lens were examined. Two sets of transgenic mice were generated, one expressing tFGFRs from the alphaA-crystallin promoter (alphaA-tFGFR), which expresses linked genes in fiber cells throughout their differentiation program, and the other expressing tFGFRs from the gammaF-crystallin promoter (gammaF-tFGFR), which expresses linked genes beginning later during their differentiation. Histological and TUNEL analyses of lenses from alphaA-tFGFR and gammaF-tFGFR transgenic mice suggest that FGFR signaling is required for both early and late fiber cell differentiation and/or survival of the terminally differentiated cells. Additionally, multilayering and increased levels of apoptosis were observed in the anterior epithelium after the onset of fiber cell abnormalities. In situ hybridizations suggest that tFGFR transgenes are not expressed at significant levels in the epithelium. Because cells derived from Rosabeta-geo26 embryos express the bacterial beta-galactosidase enzyme, which produces a blue intracellular precipitate in the presence of X-gal, they can be easily differentiated from the cells derived from nontransgenic or gammaF-tFGFR embryos. TUNEL analysis of lens sections from X-gal-stained gammaF-tFGFR/Rosabeta-geo26 aggregation chimeras reveals a high level of apoptosis in epithelial cells of both gammaF-tFGFR and Rosabeta-geo26 origin. These results confirm that the epithelial cell apoptosis in the gammaF-tFGFR lenses is not the direct result of the gammaF-tFGFR transgene and strongly suggest that the epithelial cells are dependent on the fiber cells for their survival. Thus, these results suggest that the organization and survival of the epithelial cells depend on appropriate structure and/or function of the differentiated fiber cells (Stolen, 2000).

The vertebrate lens has a distinct polarity with cuboidal epithelial cells on the anterior side and differentiated fiber cells on the posterior side. It has been proposed that the anterior-posterior polarity of the lens is imposed by factors present in the ocular media surrounding the lens (aqueous and vitreous humor). The differentiation factors have been hypothesized to be members of the fibroblast growth factor family. Though FGFs have been shown to be sufficient for induction of lens differentiation both in vivo and in vitro, they have not been demonstrated to be necessary for endogenous initiation of fiber cell differentiation. To test this possibility, transgenic mice were generated with ocular expression of secreted self-dimerizing versions of FGFR1 (FR1) and FGFR3 (FR3). Expression of FR3, but not FR1, leads to an expansion of proliferating epithelial cells from the anterior to the posterior side of the lens due to a delay in the initiation of fiber cell differentiation. This delay is most apparent postnatally and correlates with appropriate changes in expression of marker genes including p57 KIP2, Maf and Prox1. Phosphorylation of Erk1 and Erk2 was reduced in the lenses of FR3 mice, when compared with nontransgenic mice. Though differentiation was delayed in FR3 mice, the lens epithelial cells still retained their intrinsic ability to respond to FGF stimulation. Based on these results it is proposed that the initiation of lens fiber cell differentiation in mice requires FGF receptor signaling and that one of the lens differentiation signals in the vitreous humor is a ligand for FR3, and is therefore likely to be an FGF or FGF-like factor (Govindarajan, 2001).

FGF receptor and retinal and auditory differentiation

The mature vertebrate retina contains seven major cell types that develop from an apparently homogenous population of precursor cells. Clonal analyses have suggested that environmental influences play a major role in specifying retinal cell identity. Fibroblast growth factor-2 is present in the developing retina and regulates the survival, proliferation and differentiation of developing retinal cells in culture. A test was performed to see if fibroblast growth factor receptor signaling is able to bias retinal cell fate decisions in vivo. Using Xenopus embryos, fibroblast growth factor receptors were inhibited in retinal precursors by expressing a dominant negative form of the receptor, XFD. Dorsal animal blastomeres that give rise to the retina were injected with cDNA expression constructs for XFD and a control non-functional mutant receptor, D48, and the cell fates of transgene-expressing cells in the mature retina were determined. Fibroblast growth factor receptor blockade results in almost a 50% loss of photoreceptors and amacrine cells, and a concurrent 3.5-fold increase in Muller glia, suggesting a shift towards a Muller cell fate in the absence of a fibroblast growth factor receptor signal. Inhibition of non-fibroblast-growth-factor-mediated receptor signaling with a third mutant receptor, HAVO, alters cell fate in an opposite manner. These results suggest that it is the balance of fibroblast growth factor and non-fibroblast growth factor ligand signals that influences retinal cell genesis (McFarlane, 1998).

Fibroblast growth factors (FGF) 1 and 2 and their tyrosine kinase receptor (FGFR) are present throughout the adult retina. FGFs are potential mitogens, but adult retinal cells are maintained in a nonproliferative state unless the retina is damaged. A modulator of FGF signaling in normal and pathological retina was sought. A truncated FGFR1 form from rat retina generated by the use of selective polyadenylation sites was identified and sequenced. This 70-kDa form of soluble extracellular FGFR1 (SR1) is distributed mainly localized in the inner nuclear layer of the retina, whereas the full-length FGFR1 form is detected in the retinal Muller glial cells. FGF2 and FGFR1 mRNA levels greatly increase in light-induced retinal degeneration. FGFR1 is detected in the radial fibers of activated retinal Muller glial cells. In contrast, SR1 mRNA synthesis follows a biphasic pattern of down- and up-regulation, and anti-SR1 staining is intense in retinal pigmented epithelial cells. The synthesis of SR1 and FGFR1 specifically and independently regulated in normal and degenerating retina suggests that changes in the proportion of various FGFR forms may control the bioavailability of FGFs and thus their potential as neurotrophic factors. This has been demonstrated in vivo during retinal degeneration when recombinant SR1 inhibits the neurotrophic activity of exogenous FGF2 and increases damaging effects of light by inhibiting endogenous FGF. This study highlights the significance of the generation of SR1 in normal and pathological conditions (Guillonneau, 1998).

Neurons in both vertebrate and invertebrate eyes are organized in regular arrays. Although much is known about the mechanisms involved in the formation of the regular arrays of neurons found in invertebrate eyes, much less is known about the mechanisms of formation of neuronal mosaics in the vertebrate eye. The purpose of these studies was to determine the cellular mechanisms that pattern the first neurons in vertebrate retina, the retinal ganglion cells. The ganglion cells in the chick retina develop as a patterned array that spreads from the central to peripheral retina as a wave front of differentiation. The onset of ganglion cell differentiation keeps pace with overall retinal growth; however, there is no clear cell cycle synchronization at the front of differentiation of the first ganglion cells. The differentiation of ganglion cells is not dependent on signals from previously formed ganglion cells, since isolation of the peripheral retina by as much as 400 mm from the front of ganglion cell differentiation does not prevent new ganglion cells from developing. Consistent with previous studies, blocking FGF receptor activation with a specific inhibitor to the FGFRs retards the movement of the front of ganglion cell differentiation, while application of exogenous FGF1 causes the precocious development of ganglion cells in peripheral retina. These observations, taken together with those of previous studies, support a role for FGFs and FGF receptor activation in the initial development of retinal ganglion cells from the undifferentiated neuroepithelium peripheral to the expanding wave front of differentiation (McCabe, 1999).

The onset of neurogenesis in the retina occurs at the front of ganglion cell differentiation, and is controlled in part by the activation of the FGFR. The regular progression of ganglion cell differentiation from central to peripheral retina could potentially be regulated by a cell intrinsic mechanism. The early generated central retinal cells initiate ganglion cell differentiation, and as developmental time proceeds, more peripheral retinal cells reach the same state. However, the data demonstrating that the FGFR is important for the process of ganglion cell development argues that instead an extracellular signaling mechanism is more likely to be important for this process. The data indicate a role for the FGF receptor family of tyrosine kinases in the initial differentiation of ganglion cells at the front of differentiation: blocking FGFR activation with SU5402 inhibits ganglion cell formation from the undifferentiated neuroepithelium. Although the application of SU5402 to the explants could be delaying rather than blocking ganglion cell differentiation, it is clear that FGFR activation plays an important part in the movement of the front of ganglion cell differentiation. Of the four FGF receptors, the data from this and other studies indicate that FGFR1 is most likely to mediate the differentiation of ganglion cells. In the chick embryo, FGFR1 is expressed in the neuroepithelium of the early optic vesicle. Soon after the formation of the optic cup, FGFR1 is expressed in a central to peripheral gradient in the retina, with the strongest expression peripherally, in a pattern consistent with a role for regulating the onset of ganglion cell differentiation. By stage 24 (E4), the gradient of FGFR1 has become more distinct, although FGFR1 is still expressed in the central retina at low levels (McCabe, 1999).

Since activation of the FGF receptor is important for the progression of the front of ganglion cell differentiation, it is important to identify whether one of the previously identified ligands for these receptors is expressed in the retina when the ganglion cells are developing. FGF ligand isoforms that are expressed in the eye include FGF1, FGF2, FGF8, FGF11, FGF12, FGF14 and FGF15. The data indicate that the ligand necessary for the progression of ganglion cell differentiation is most likely located in the peripheral retina, since the central retina is not necessary for this process. Two FGFs have been shown to be localized in peripheral retina in some vertebrate species: FGF1 and FGF15. However, it is also possible that an additional member of this very large family may also be localized in this region, and future studies will be necessary to determine which of these potential ligands are critical for ganglion cell differentiation (McCabe, 1999).

Several studies suggest fibroblast growth factor receptor 3 (FGFR3) plays a role in the development of the auditory epithelium in mammals. A study was undertaken of FGFR3 in the developing and mature chicken inner ear and during regeneration of this epithelium to determine whether FGFR3 shows a similar pattern of expression in birds. FGFR3 mRNA is highly expressed in most support cells in the mature chick basilar papilla but not in vestibular organs of the chick. The gene is expressed early in the development of the basilar papilla. Gentamicin treatment sufficient to destroy hair cells in the basilar papilla causes a rapid, transient downregulation of FGFR3 mRNA in the region of damage. In the initial stages of hair cell regeneration, the support cells that reenter the mitotic cycle in the basilar papilla do not express detectable levels of FGFR3 mRNA. However, once the hair cells have regenerated in this region, the levels of FGFR3 mRNA and protein expression rapidly return to approximate those in the undamaged epithelium. These results indicate that FGFR3 expression changes after drug-induced hair cell damage to the basilar papilla in a way opposite that found in the mammalian cochlea and may be involved in regulating the proliferation of support cells (Bermingham-McDonogh, 2001).

The mammalian auditory sensory epithelium, the organ of Corti, comprises the hair cells and supporting cells that are pivotal for hearing function. The origin and development of their precursors are poorly understood. Loss-of-function mutations in mouse fibroblast growth factor receptor 1 (Fgfr1) cause a dose-dependent disruption of the organ of Corti. Full inactivation of Fgfr1 in the inner ear epithelium by Foxg1-Cre-mediated deletion leads to an 85% reduction in the number of auditory hair cells. The primary cause appears to be reduced precursor cell proliferation in the early cochlear duct. Thus, during development, FGFR1 is required for the generation of the precursor pool, which gives rise to the auditory sensory epithelium. These data also suggest that FGFR1 might have a distinct later role in intercellular signaling within the differentiating auditory sensory epithelium (Pirvola, 2002).

One of the most striking aspects of the cellular pattern within the sensory epithelium of the mammalian cochlea is the presence of two rows of pillar cells in the region between the single row of inner hair cells and the first row of outer hair cells. The factors that regulate pillar cell development have not been determined; however, previous results have suggested a key role for fibroblast growth factor receptor 3 (FGFR3). To examine the specific effects of FGFR3 on pillar cell development, receptor activation was inhibited in embryonic cochlear explant cultures. Results indicate that differentiation of pillar cells is dependent on continuous activation of FGFR3. Moreover, transient inhibition of FGFR3 does not inhibit the pillar cell fate permanently, because reactivation of FGFR3 results in the resumption of pillar cell differentiation. The effects of increased FGFR3 activation were determined by exposing cochlear explants to FGF2, a strong ligand for several FGF receptors. Treatment with FGF2 led to a significant increase in the number of pillar cells and to a small increase in the number of inner hair cells. These effects are not dependent on cellular proliferation, suggesting that additional pillar cells and inner hair cells are a result of increased recruitment into the prosensory domain. These results indicate that FGF signaling plays a critical role in the commitment and differentiation of pillar cells. Moreover, the position of the pillar cells appears to be determined by the activation of FGFR3 in a subset of the progenitor cells that initially express this receptor (Mueller, 2002).

Molecular analysis of vertebrate eye development has been hampered by the availability of sequences that can selectively direct gene expression in the developing eye. Regulatory sequences of the Xenopus laevis Rx1A gene (homolog of Drosophila Rx homeodomain transcription factor) can direct gene expression in the retinal progenitor cells. These sequences have been used to investigate the role of Fibroblast Growth Factor (FGF) signaling in the development of retinal cell types. FGFs are signaling molecules that are crucial for correct patterning of the embryo and play important roles in the development of several embryonic tissues. FGFs and their receptors are expressed in the developing retina, and FGF receptor-mediated signaling has been implicated in the specification and survival of retinal cell types. The role of FGF signaling mediated by FGF receptor 4a has been investigated in the development of retinal cell types in Xenopus laevis. For this purpose, transgenic Xenopus tadpoles were made in which the dominant-negative FGFR4a (DeltaFGFR4a) coding region was linked to the newly characterized regulatory sequences of the Xrx1A gene. The expression of DeltaFGFR4a in retinal progenitor cells results in abnormal retinal development. The retinas of transgenic animals expressing DeltaFGFR4a show disorganized cell layering and specifically lack photoreceptor cells. These experiments show that FGFR4a-mediated FGF signaling is necessary for the correct specification of retinal cell types. Furthermore, they demonstrate that constructs using Xrx1A regulatory sequences are excellent tools with which to study the developmental processes involved in retinal formation (Zhang, 2003).

Dorsoventral patterning of the Xenopus eye: a collaboration of Retinoid, Hedgehog and FGF receptor signaling

In the developing spinal cord and telencephalon, ventral patterning involves the interplay of Hedgehog (Hh), Retinoic Acid (RA) and Fibroblast Growth Factor (FGF) signaling. In the eye, ventral specification involves Hh signaling, but the roles of RA and FGF signaling are less clear. By overexpression assays in Xenopus embryos, it was found that both RA and FGF receptor (FGFR) signaling ventralize the eye, by expanding optic stalk and ventral retina, and repressing dorsal retina character. Co-overexpression experiments show that RA and FGFR can collaborate with Hh signaling and reinforce its ventralizing activity. In loss-of-function experiments, a strong eye dorsalization is observed after triple inhibition of Hh, RA and FGFR signaling, while weaker effects are obtained by inhibiting only one or two of these pathways. These results suggest that the ventral regionalization of the eye is specified by interactions of Hh, RA and FGFR signaling. It is argued that similar mechanisms might control ventral neural patterning throughout the central nervous system (Lupo, 2005).

Dorsoventral (DV) patterning of the vertebrate eye underlies important properties of the visual system. First, it controls subdivision of the eye into optic stalk (OS) and retina, which form from the ventromedial and the dorsolateral parts of the optic vesicle, respectively. Second, the retina itself is patterned along the DV axis. A landmark of retina DV polarity is the choroid fissure at the ventral pole of the retina. Retinal neurogenesis is initiated in this region and then progresses to the dorsal retina (DR). DV asymmetries also exist in the distribution of differentiated cell types within the retina. Finally, ganglion cells located in the dorsal retina and the ventral retina (VR) send their axons to the lateral and medial optic tectum, respectively, creating an inverted topographic map of retinotectal projections (Lupo, 2005).

A model of ventral eye specification is proposed that involves interactions among RA, Hh and FGFR signaling pathways. According to this model, high levels of Hh and FGFR signaling interact with low levels of RA signaling to specify the OS by repressing retina-determination genes such as Pax6, and promoting the expression of Vax1 and Pax2. By contrast, high levels of RA act in concert with lower levels of Hh and FGFR signaling to specify the VR by repressing DR-specific genes such as ET and by inducing the expression of Vax2 in the presence of Pax6, but not Vax1 and Pax2. BMP signaling specifies DR regions by repressing Vax2 and inducing ET and other members of the Tbx gene family, such as Tbx5. In vivo, ventrodorsal (ventral high) gradients of Hh and FGFR signaling may be created by diffusion of Hh and FGF signals from their sources in the anteromedial neural plate. The regulation of RA gradients is more complex and the localization of different anabolic and catabolic enzymes needs to be considered. However, early expression of Raldh2 and Raldh3 appears to be localized close to the presumptive ventral eye, and the mediolateral gradient (lateral high) of Raldh2 expression in the ANR may contribute to create higher RA levels in the VR compared with the OS region (Lupo, 2005).

Recent studies have shown that ventral patterning in the spinal cord and the telencephalon involves interactions between Hh, RA and FGF signaling pathways. In the spinal cord, motoneurons and V3, V2 ventral interneurons originate from the ventral neural tube, while V1 and V0 ventral interneurons originate from more intermediate regions. Hh signals from the notochord and floorplate are thought to specify the progenitor domain of motoneurons, V3 and V2 interneurons, while RA signaling is crucial for the specification of V1 and V0 interneurons. In addition, although FGF signaling appears to function as a general repressor of ventral neural patterning, RA and FGF in combination can efficiently induce motoneuron progenitors both in explants and in vivo (Lupo, 2005).

In the telencephalon, the medial ganglionic eminence (MGE) originates from the ventral part of the telencephalic vesicle, while the lateral ganglionic eminence (LGE) originates from a more intermediate region. Hh signaling is involved in the specification of the MGE, while RA signaling appears to play a crucial role in the specification of the LGE. In addition, FGF signaling is involved in the specification of ventral, but not intermediate, telencephalic fates (Lupo, 2005).

In the developing eye, cells located more ventrally in the anlage give rise to the OS, while the VR originates from a more intermediate region. Hh and FGFR signaling play a crucial role in OS specification, although low levels of RA signaling may also be involved. Moreover, RA signaling could control specification of the VR in collaboration with low levels of Hh and possibly FGFR signaling (Lupo, 2005).

Similar mechanisms of ventral specification involving Hh, RA and FGFR signaling pathways appear to be at least partially conserved in different CNS regions. Several questions remain to be addressed concerning the precise role and the mechanism of action of these signaling systems. Clearly, DV patterning of the vertebrate CNS is a complex process, and the eye, because of its distinct regional composition, its finely graded topography and its experimental accessibility, is an exciting model with which to study how different signaling pathways interact to execute specific developmental programs (Lupo, 2005).

FGF receptor, neural induction, patterning and differentiation

Neural induction constitutes the first step in the generation of the vertebrate nervous system from embryonic ectoderm. Work with Xenopus ectodermal explants has suggested that epidermis is induced by BMP signals, whereas neural fates arise by default following BMP inhibition. In amniotes and ascidians, however, BMP inhibition does not appear to be sufficient for neural fate acquisition, which is initiated by FGF signalling. The roles of the BMP and FGF pathways during neural induction in Xenopus have been reevaluated. Ectopic BMP activity converts the neural plate into epidermis, confirming that this pathway must be inhibited during neural induction in vivo. Conversely, inhibition of BMP, or of its intracellular effector SMAD1 in the non-neural ectoderm leads to epidermis suppression. In no instances, however, is BMP/SMAD1 inhibition sufficient to elicit neural induction in ventral ectoderm. By contrast, neural specification occurs when weak eFGF or low ras signalling are combined with BMP inhibition. Using all available antimorphic FGF receptors (FGFR), as well as the pharmacological FGFR inhibitor SU5402, it was demonstrated that pre-gastrula FGF signalling is required in the ectoderm for the emergence of neural fates. Finally, although the FGF pathway contributes to BMP inhibition, as in other model systems, it is also essential for neural induction in vivo and in animal caps in a manner that cannot be accounted for by simple BMP inhibition. Taken together, these results reveal that in contrast to predictions from the default model, BMP inhibition is required but not sufficient for neural induction in vivo. This work contributes to the emergence of a model whereby FGF functions as a conserved initiator of neural specification among chordates (Delaune, 2005).

Cell adhesion molecules (CAMs) are good candidates for the positive cues that promote and/or guide axons out of the developing mammalian retina. The activation of a tyrosine kinase-phospholipase C gamma cascade is important for the neurite outgrowth responses stimulated by NCAM (Drosophila homolog FAS2), N-cadherin (see Drosophila Cadherin-N) and L1 (Drosophila homolog: Neuroglian). It is thought that the neurite growth response stimulated by these CAMs is mediated by activation of the fibroblast growth factor receptor FGFR in neurons. For example, fibroblast growth factor receptor function is required for the orderly projection of ganglion cells to the optic fissure. FGFR intracellular domain recruits and activates phospholipase C gamma via interactions of PLC gamma SH2 domain with the activated receptor. The key events downstream from activation of PLC gamma are the generation of diacylglycerol and the conversion of diacylglycerol to arachidonic acid via DAG lipase activity. Subsequently AA, interacting with calcium channels, induces an increased influx of calcium into neurons. The CAMs are able to interact with the FGFR extracellular domains, in cis (adjacently on the same cell membrane), via conserved interaction motifs, thus recruiting FGFR to sites of homophilic CAD interaction to engender FGFR activation, and thus promoting axonal growth (Doherty, 1996 and references).

Activation of cell adhesion molecules NCAM (Drosophila homolog FAS2) and L1 can lead to phosphorylation of the fibroblast growth factor receptor (FGFR). Both phosphorylation and the neurite outgrowth response stimulated by these CAMs are lost when a kinase-deleted, dominant negative form of murine FGFR1 is expressed in PC12 cells. Cerebellar neurons, isolated from transgenic mice that express the dominant negative FGFR, have lost their ability to respond to NCAM, N-cadherin and L1. A peptide inhibitor of phospholipase C gammaresults in the inhibition of neurite outgrowth normally stimulated by FGF. The same inhibitor also blocks neurite outgrowth stimulated by the CAMS. It is concluded that activation of FGFR is both necessary and sufficient to account for the ability of these CAMs to stimulate axonal growth, and PLCgamma is a key downstream effector of this response (Saffell, 1997).

Fibroblast growth factor receptor-1 (FGFR-1) is a membrane-spanning tyrosine kinase that serves as a high-affinity receptor for fibroblast growth factors. It has recently been shown that FGFR-1 mutant embryos die during gastrulation displaying severe growth retardation and defective mesodermal structures. This early lethality has obscured functions of FGFR-1 that might occur later in development. To circumvent these embryonic defects, chimeras were generated by injecting FGFR-1-deficient (R1 +/-) embryonic stem (ES) cells into wild-type blastocysts. It was found that the fgfr-1 gene plays an important role after gastrulation, and that it acts in a cell-autonomous fashion. Embryos with a high contribution of R1-/- cells replicate the FGFR-1 null phenotype and die during gastrulation. In contrast, the majority of embryos with a low contribution of R1-/- cells complete gastrulation and display malformations of posterior structures at later stages of embryogenesis. These abnormalities include truncation of embryonic structures, limb bud malformation, partial duplication of the neural tube, tail distortion, and spina bifida caused by the amplification of neural tissue in the posterior portion of the spinal cord. Thus, FGFR-1 plays a role in neurulation, suggesting that there may be a connection between FGFR-1-mediated signal pathways and neural tube defects, the most common malformations in the human central nervous system (Deng, 1997).

Epidermal growth factor induces PC12 cell differentiation in the presence of the protein kinase inhibitor K-252a. K-252a blocks the actions of nerve growth factor and other neurotrophins and, at lower concentrations, selectively potentiates neurotrophin-3 actions. K-252a, enhances epidermal growth factor (EGF)-and basic fibroblast growth factor (BFGF)-induced neurite outgrowth of PC12 cells at higher concentrations than required for neurotrophin inhibition. In parallel, tyrosine phosphorylation of extracellular signal-regulated kinases (Erks)(Drosophila homolog: Rolled/MAP kinase) elicited by EGF or bFGF is also increased in the presence of K-252a, and this signal was prolonged for 6 h. EGF- and bFGF-induced phosphorylation of phospholipase C-gamma 1 were not changed. The effect of K-252a on Erks is resistant to chronic treatment with phorbol ester, indicating that protein kinase C is not involved in this potentiation. Although K-252a alone does not induce neurite outgrowth or tyrosine phosphorylation of Erks or phospholipase C-gamma 1, this compound alone stimulates phosphatidylinositol hydrolysis. These findings identify activities of K-252a besides the direct interaction with neurotrophin receptors and suggest that a K-252a-sensitive protein kinase or phosphatase might be involved in signal transduction of EGF and bFGF. These results are also compatible with the hypothesis that sustained activation of Erks may be important in PC12 differentiation (Isono, 1994).

Basic fibroblast growth factor (bFGF) plays an important role in development of the central nervous system and is neurotropic for a variety of neurons. bFGF is neurotropic for GT1 GnRH neuronal cells which express functional FGF receptors (FGFRs). The GT1 cell lines generated by genetically targeted tumorigenesis display highly differentiated properties of GnRH neurons. Addition of 2 and 10 ng/ml bFGF increases neurite outgrowth of GT1-7 cells and results in a significant increase of GT1 cell survival in serum-free medium. GT1 cells express FGFRs 1 and 3 but not 2. Occupancy of FGFRs with 10 ng/ml bFGF stimulates the sustained tyrosine phosphorylation of both the 42- and 44-kilodalton mitogen-activated protein kinases (MAPKs) for up to 6 h. GT1-1 and GT1-7 cells also express messenger RNA for bFGF, although the level of bioactive bFGF synthesized by GT1 cells appears suboptimal because GT1 cells can further respond to exogenously added bFGF. Thus, bFGF is a neurotropic factor in GT1 GnRh neuronal cell lines, raising the possibility that bFGF may play a role in the neurobiology of GnRH neurons (Tsai, 1995).

The proliferation, migration, survival, and differentiation of oligodendrocyte progenitor cells, precursors to myelin-forming oligodendrocytes in the CNS, are controlled by a number of polypeptide growth factors in vitro. The requirement and roles for individual factors in vivo, however, are primarily unknown. A cell transplantation approach was used to examine the role of fibroblast growth factor (FGF) in oligodendrocyte development in vivo. A dominant-negative version of the FGF receptor-1 transgene was introduced into oligodendrocyte progenitors in vitro, generating cells that are nonresponsive to FGF but responsive to other mitogens. When transplanted into the brains of neonatal rats, mutant cells are unable to migrate and remain within the ventricles. These results suggest a role for FGF signaling in establishing a motile phenotype for oligodendrocyte progenitor cell migration in vivo and illustrate the utility of a somatic cell mutagenesis approach for the study of gene function during CNS development in vivo (Osterhout, 1997).

The expression pattern of the fibroblast growth factor receptor Fgf-R1, R2 and R3 genes was studied in the chicken spinal cord using in situ hybridization (ISH). Unlike Fgf-R1, which is widely expressed in motoneurons, Fgf-R3 is expressed in a subset of motoneurons in the medial subdivision of the median motor column that also expresses Islet-1 and Lim-3. The motoneuron identity of the labelled cells was confirmed by double ISH and by single cell RT-PCR. Interestingly, E3.5 spinal cord motoneurons do not express Fgf-R3, suggesting the expression of Fgf-R3 in motoneurons begins with axonal growth (Philippe, 1998).

Fibroblast growth factor (FGF) has been proposed to be involved in the specification and patterning of the developing vertebrate nervous system. There is conflicting evidence, however, concerning the requirement for FGF signaling in these processes. To provide insight into the signaling mechanisms that are important for neural induction and anterior-posterior neural patterning, the dominant negative Ras mutant, N17Ras, was employed, in addition to a truncated FGF receptor (XFD). Both N17Ras and XFD, when expressed in Xenopus laevis animal cap ectoderm, inhibit the ability of FGF to generate neural pattern. They also block induction of posterior neural tissue by XBF2 and XMeis3. However, neither XFD nor N17Ras inhibits noggin, neurogenin, or XBF2 induction of anterior neural markers. MAP kinase activation has been proposed to be necessary for neural induction, yet N17Ras inhibits the phosphorylation of MAP kinase that usually follows explantation of explants. In whole embryos, Ras-mediated FGF signaling is critical for the formation of posterior neural tissues but is dispensable for neural induction (Ribisi, 2000).

Posterior mesoderm tissue induces midbrain and hindbrain fates from prospective forebrain, an activity that is mimicked in explant culture by bFGF. Treatment of early gastrula age animal cap ectoderm with bFGF protein induces the expression of the spinal cord marker hoxB9. Late gastrula-age (stage 11) animal cap ectoderm treated with bFGF expresses the midbrain and hindbrain marker genes en2 and krox20, in addition to hoxB9, and the forebrain marker otx2 is not induced. The combination of somite tissue with animal caps of gastrula age (stage 10.5) induces the expression of hindbrain-specific genes and low levels of spinal cord-specific genes in the animal cap tissue and this induction is partially sensitive to XFD. Keller explants faithfully recapitulate the A-P distribution of neural markers observed in the whole embryo: blocking FGF signaling using XFD eliminates posterior neural development in Keller explants. The claim that FGF signaling is required for the formation of posterior neural tissue is supported by the results of explant assays. The induction of posterior neural markers requires FGF and Ras signaling. Animal caps do not express posterior neural markers in response to either XBF2 or XMeis3 when either FGF or Ras signaling is blocked. In addition, when MAPK activation is directly inhibited by MAP kinase phosphatase, the ability of XMeis3 to induce the expression of posterior neural markers is greatly curtailed (Ribisi, 2000 and references therein).

The role of fibroblast growth factors (FGFs) in neural induction is controversial. Although FGF signaling has been implicated in early neural induction, a late role for FGFs in neural development is not well established. Indeed, it is thought that FGFs induce a precursor cell fate but are not able to induce neuronal differentiation or late neural markers. It is also not known whether the same or distinct FGFs and FGF receptors (FGFRs) mediate the effects on mesoderm and neural development. Xenopus embryos expressing ectopic FGF-8 develop an abundance of ectopic neurons that extend to the ventral, non-neural ectoderm, but show no ectopic or enhanced notochord or somitic markers. FGF-8 inhibits the expression of an early mesoderm marker, Xbra, in contrast to eFGF, which induces ectopic Xbra robustly and neuronal differentiation weakly. The effect of FGF-8 on neurogenesis is blocked by dominant-negative FGFR-4a (deltaXFGFR-4a). Endogenous neurogenesis is also blocked by deltaXFGFR-4a and less efficiently by dominant-negative FGFR-1 (XFD), suggesting that it depends preferentially on signaling through FGFR-4a. The results suggest that FGF-8 and FGFR-4a signaling promotes neurogenesis and, unlike other FGFs, FGF-8 interferes with mesoderm induction. Thus, different FGFs show specificity for mesoderm induction versus neurogenesis and this may be mediated, at least in part, by the use of distinct receptors (Hardcastle, 2000).

During development, the embryonic telencephalon is patterned into different areas that give rise to distinct adult brain structures. Several secreted signaling molecules are expressed at putative signaling centers in the early telencephalon. In particular, Fgf8 is expressed at the anterior end of the telencephalon and is hypothesized to pattern it along the anteroposterior (AP) axis. Using a CRE/loxP genetic approach to disrupt genes in the telencephalon, the role of FGF signaling directly has been assessed in vivo by abolishing expression of the FGF receptor Fgfr1. In the Fgfr1-deficient telencephalon, AP patterning is largely normal. However, morphological defects are observed at the anterior end of the telencephalon. Most notably, the olfactory bulbs do not form normally. Examination of the proliferation state of anterior telencephalic cells supports a model for olfactory bulb formation in which an FGF-dependent decrease in proliferation is required for initial bulb evagination. Together the results demonstrate an essential role for Fgfr1 in patterning and morphogenesis of the telencephalon (Hébert, 2003).

The postnatal central nervous system contains many scattered cells that express fibroblast growth factor receptor 3 transcripts (Fgfr3). They first appear in the ventricular zone (VZ) of the embryonic spinal cord in mid-gestation and then distribute into both grey and white matter, suggesting that they are glial cells, not neurons. The Fgfr3+ cells are interspersed with, but distinct from, platelet-derived growth factor receptor alpha (Pdgfra)-positive oligodendrocyte progenitors. This fits with the observation that Fgfr3 expression is preferentially excluded from the pMN domain of the ventral VZ where Pdgfra+ oligodendrocyte progenitors (and motoneurons) originate. Many glial fibrillary acidic protein (Gfap)-positive astrocytes co-express Fgfr3 in vitro and in vivo. Fgfr3+ cells within and outside the VZ also express the astroglial marker glutamine synthetase (Glns). It is concluded that (1) Fgfr3 marks astrocytes and their neuroepithelial precursors in the developing CNS and (2) astrocytes and oligodendrocytes originate in complementary domains of the VZ. Production of astrocytes from cultured neuroepithelial cells is hedgehog independent, whereas oligodendrocyte development requires hedgehog signalling, adding further support to the idea that astrocytes and oligodendrocytes can develop independently. In addition, mice with a targeted deletion in the Fgfr3 locus strongly upregulate Gfap in grey matter (protoplasmic) astrocytes, implying that signalling through Fgfr3 normally represses Gfap expression in vivo (Pringle, 2003).

Sonic hedgehog (SHH) and fibroblast growth factor 2 (FGF2) can both induce neocortical precursors to express the transcription factor OLIG2 and generate oligodendrocyte progenitors (OLPs) in culture. The activity of FGF2 is unaffected by cyclopamine, which blocks Hedgehog signalling, demonstrating that the FGF pathway to OLP production is Hedgehog independent. Unexpectedly, SHH-mediated OLP induction is blocked by PD173074, a selective inhibitor of FGF receptor (FGFR) tyrosine kinase. SHH activity also depends on mitogen-activated protein kinase (MAPK) but SHH does not itself activate MAPK. Instead, constitutive activity of FGFR maintains a basal level of phosphorylated MAPK that is absolutely required for the OLIG2- and OLP-inducing activities of SHH. Stimulating the MAPK pathway with a retrovirus encoding constitutively active RAS shows that the requirement for MAPK is cell-autonomous, i.e. MAPK is needed together with SHH signalling in the cells that become OLPs. It seems likely that SHH needs a basal level of active MAPK in order to function, and that constitutive FGFR activity in cortical cultures provides the necessary stimulus (Kessaris, 2004).

Recent findings support a model for neocortical area formation in which neocortical progenitor cells become patterned by extracellular signals to generate a protomap of progenitor cell areas that in turn generate area-specific neurons. The protomap is thought to be underpinned by spatial differences in progenitor cell identity that are reflected at the transcriptional level. The nature and composition of the protomap was systematically investigated by genomic analyses of spatial and temporal neocortical progenitor cell gene expression. No gene expression evidence was found for progenitor cell organisation into domains or compartments: instead rostrocaudal gradients of gene expression were found across the entire neocortex. Given the role of Fgf signalling in rostrocaudal neocortical patterning, an in vivo global analysis of cortical gene expression was carried out in Fgfr1 mutant mice, identifying consistent alterations in the expression of candidate protomap elements. One such gene, Mest (coding for a enzyme with a predicted alpha/beta hydrolase fold), was predicted by those studies to be a direct target of Fgf8 signalling and to be involved in setting up, rather than implementing, the progenitor cell protomap. In support of this, Mest was confirmed to be a direct transcriptional target of Fgf8-regulated signalling in vitro. Functional studies demonstrate that this gene has a role in establishing patterned gene expression in the developing neocortex, potentially by acting as a negative regulator of the Fgf8-controlled patterning system. Although the cellular function of Mest/Peg1 is unknown, this protein is highly conserved throughout its length in vertebrates. Notably, an orthologous protein cannot be found in the Drosophila or C. elegans genomes, suggesting that this particular protein is vertebrate specific (Sansom, 2005).

Syndecan-4 (Syn4) is a heparan sulphate proteoglycan that is able to bind to some growth factors, including FGF, and can control cell migration. This study describes a new role for Syn4 in neural induction in Xenopus. Syn4 is expressed in dorsal ectoderm and becomes restricted to the neural plate. Knockdown with antisense morpholino oligonucleotides reveals that Syn4 is required for the expression of neural markers in the neural plate and in neuralised animal caps. Injection of Syn4 mRNA induces the cell-autonomous expression of neural, but not mesodermal, markers. Two parallel pathways are involved in the neuralising activity of Syn4: FGF/ERK, which is sensitive to dominant-negative FGF receptor and to the inhibitors SU5402 and U0126, and a PKC pathway, which is dependent on the intracellular domain of Syn4. Neural induction by Syn4 through the PKC pathway requires inhibition of PKCdelta and activation of PKCalpha. PKCalpha inhibits Rac GTPase and c-Jun is a target of Rac. These findings might account for previous reports implicating PKC in neural induction and suggest a link between FGF and PKC signalling pathways during neural induction (Kuriyama, 2009).

Syn4 modulates FGF signalling through its extracellular domain (containing the GAG-binding region, which will present heparin sulphates to which FGF is expected to bind) and by an effect on the transduction of intracellular signals. The data support the idea that FGF is required for neural induction and that Syn4 is a likely modulator, by showing that the inhibition of FGF receptor and of MAPK activity impair neural induction by Syn4. Syn4 could act as a co-receptor of the FGF receptor or as a presenter of the FGF ligand, through binding of FGF to the GAG side-chains, to facilitate the activation of FGF receptor (Kuriyama, 2009).

However, Syn4 also plays a separate role in neural induction involving PKC. It is proposed that this involves inhibition of PKC{delta} and activation of PKC{alpha}, and that PKC{alpha} is an inhibitor of the small GTPase Rac. Since the BMP-inhibiting effects of FGF act through MAPK, this pathway could account for the BMP-inhibition-independent role of FGF signalling in neural induction. Rac is a well-known regulator of cell migration that acts by controlling actin polymerisation, but has not previously been implicated in neural induction. Evidence that Rac can control JNK activity suggested the hypothesis that Syn4/PKC{alpha} might inhibit Rac activity by an increase in AP-1 (c-Fos/c-Jun) activity that is mediated through inhibition of JNK (Kuriyama, 2009).

PKC{alpha} has never been connected with the signalling pathways now known to be involved in neural induction. It was originally shown that PKC{alpha} is activated and translocated to the membrane during neural induction, and it was suggested that this is required to confer neural competence on the ectoderm. This study has confirmed and extended these observations by showing that expression of PKC{alpha} in ventral ectoderm or in animal caps can act as a neuralising signal and that PKC{alpha} activity is regulated by interactions with Syn4 and PKC{delta}. PKC{delta} appears to work as a repressor of PKC{alpha}, whereas Syn4 appears to be required for PKC{alpha} activity; however, it was also shown that PKC{alpha} is required for the neuralising activity of Syn4. Thus, this finding allows proposal of a link between the PKC and FGF pathways, both of which have been identified as being involved in neural induction (Kuriyama, 2009).

These observations have parallels in studies of migrating cells. Syn4 interacts with PIP2, and this stabilises the oligomeric structure of Syn4 and promotes the association of PKC{alpha} and Syn4; the catalytic domain of PKC{alpha} binds to the cytoplasmic domain of Syn4, and PKC{alpha} is 'superactivated'. This interaction between PKC{alpha} and Syn4 provides a satisfactory explanation for the observation that neural induction by Syn4 requires PKC{alpha} and vice versa. In addition, during cell migration, PKC{delta} phosphorylates Syn4, decreases its affinity for PIP2 and abolishes its capacity to activate PKC{alpha}. This study has found a similar negative regulation between PKC{alpha} and PKC{delta} during early neural plate development (Kuriyama, 2009).

The mechanisms underlying the generation of neural cell diversity are the subject of intense investigation, which has highlighted the involvement of different signalling molecules including Shh, BMP and Wnt. By contrast, relatively little is known about FGF in this process. This study has identified an FGF-receptor-dependent pathway in zebrafish hindbrain neural progenitors that give rise to somatic motoneurons, oligodendrocyte progenitors and differentiating astroglia. Using a combination of chemical and genetic approaches to conditionally inactivate FGF-receptor signalling, the role of this pathway was investigated. It was shown that FGF-receptor signalling is not essential for the survival or maintenance of hindbrain neural progenitors but controls their fate by coordinately regulating key transcription factors. First, by cooperating with Shh, FGF-receptor signalling controls the expression of olig2, a patterning gene essential for the specification of somatic motoneurons and oligodendrocytes. Second, FGF-receptor signalling controls the development of both oligodendrocyte progenitors and astroglia through the regulation of sox9, a gliogenic transcription factor the function of which is conserved in the zebrafish hindbrain. Overall, these results reveal a mechanism of FGF in the control of neural cell diversity in the hindbrain (Esain, 2010).

The olfactory sensory epithelium and the respiratory epithelium are derived from the olfactory placode. However, the molecular mechanisms regulating the differential specification of the sensory and the respiratory epithelium have remained undefined. To address this issue, first, Msx1/2 and Id3 were identified as markers for respiratory epithelial cells, by performing quail chick transplantation studies. Next, chick explant and intact chick embryo assays of sensory/respiratory epithelial cell differentiation were established and two mice mutants deleted of Bmpr1a;Bmpr1b or Fgfr1;Fgfr2 in the olfactory placode were analyzed. In this study, evidence is provided that in both chick and mouse, Bmp signals promote respiratory epithelial character, whereas Fgf signals are required for the generation of sensory epithelial cells. Moreover, olfactory placodal cells can switch between sensory and respiratory epithelial cell fates in response to Fgf and Bmp activity, respectively. These results provide evidence that Fgf activity suppresses and restricts the ability of Bmp signals to induce respiratory cell fate in the nasal epithelium. In addition, in both chick and mouse the lack of Bmp or Fgf activity results in disturbed placodal invagination; however, the fate of cells in the remaining olfactory epithelium is independent of morphological movements related to invagination. In summary, a conserved mechanism in amniotes is presented in which Bmp and Fgf signals act in an opposing manner to regulate the respiratory versus sensory epithelial cell fate decision (Maier, 2010).

FGF receptor and axon outgrowth

Wiring of the nervous system requires that axons navigate to their targets and maintain their correct positions in axon fascicles after termination of axon outgrowth. The C. elegans fibroblast growth factor receptor (FGFR), EGL-15, affects both processes in fundamentally distinct manners. FGF-dependent activation of the EGL-15 tyrosine kinase and subsequently the GTPase LET-60/ras is required within epidermal cells, the substratum for most outgrowing axon, for appropriate outgrowth of specific axon classes to their target area. In contrast, genetic elimination of the FGFR isoform EGL-15(5A), defined by the inclusion of an alternative extracellular interimmunoglobulin domain, has no consequence for axon outgrowth but leads to a failure to postembryonically maintain axon position within defined axon fascicles. An engineered, secreted form of EGL-15(5A) containing only its ectodomain is sufficient for maintenance of axon position, thus providing novel insights into receptor tyrosine kinase function and the process of maintaining axon position (Bülow, 2004).

Table of contents

FGF receptor 1 continued: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.