Interactive Fly, Drosophila

FGF receptor in C. elegans

Myogenesis in vertebrate myocytes is promoted by activation of the phosphatidyl-inositol 3'-kinase (PI3 kinase) pathway and inhibited by fibroblast growth factor (FGF) signaling. Hyperactivation of the Caenorhabditis elegans FGF receptor, EGL-15, similarly inhibits the differentiation of the hermaphrodite sex muscles. Activation of the PI3 kinase signaling pathway can partially suppress this differentiation defect, mimicking the antagonistic relationship between these two pathways known to influence vertebrate myogenesis. When ectopically expressed in body wall muscle precursor cells, hyperactivated EGL-15 can also interfere with the proper development of the body wall musculature. Hyperactivation of EGL-15 has also revealed additional effects on a number of fundamental processes within the postembryonic muscle lineage, such as cell division polarity. These studies provide important in vivo insights into the contribution of FGF signaling events to myogenesis (Sasson, 2004).

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

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

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

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

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

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

FGF receptor and embryonic stem cells

Embryonic stem (ES) cells can be derived and propagated from multiple strains of mouse and rat through application of small-molecule inhibitors of the fibroblast growth factor (FGF)/Erk pathway and of glycogen synthase kinase 3. These conditions shield pluripotent cells from differentiation-inducing stimuli. This study investigated the effect of these inhibitors on the development of pluripotent epiblast in intact pre-implantation embryos. Blockade of Erk signalling from the 8-cell stage was found not impede blastocyst formation but suppresses development of the hypoblast. The size of the inner cell mass (ICM) compartment is not reduced, however. Throughout the ICM, the epiblast-specific marker Nanog is expressed, and in XX embryos epigenetic silencing of the paternal X chromosome is erased. Epiblast identity and pluripotency were confirmed by contribution to chimaeras with germline transmission. These observations indicate that segregation of hypoblast from the bipotent ICM is dependent on FGF/Erk signalling and that in the absence of this signal, the entire ICM can acquire pluripotency. Furthermore, the epiblast does not require paracrine support from the hypoblast. Thus, naive epiblast and ES cells are in a similar ground state, with an autonomous capacity for survival and replication, and high vulnerability to Erk signalling. The relationship between naive epiblast and ES cells was probed directly. Dissociated ICM cells from freshly harvested late blastocysts gave rise to up to 12 ES cell clones per embryo when plated in the presence of inhibitors. It is proposed that ES cells are not a tissue culture creation, but are essentially identical to pre-implantation epiblast cells (Nichols, 2009).

FGF receptor, gastrulation and axis formation

The tadpole larva of an ascidian (phylum Urochordata) develops 40 notochord cells in the center of its tail. Most of the notochord cells originate from the A-line precursors, among which inductive interactions are required for the subsequent differentiation of notochord. The presumptive-endoderm blastomeres or presumptive-notochord blastomeres themselves are inducers of notochord formation. Notochord induction takes place during the 32-cell stage. In amphibia, mesoderm induction is thought to be mediated by several growth factors, for example, activins and basic fibroblast growth factor (bFGF). In the ascidian, Halocynthia roretzi, treatment with bFGF of presumptive-notochord blastomeres that had been isolated at the early 32-cell stage promotes the formation of notochord at a low concentration of bFGF, while activin fails to induce notochord differentiation. The effect of bFGF reaches a maximum at the end of the 32-cell stage and rapidly fades at the beginning of the subsequent cleavage, the time for full induction of notochord being at least 20 minutes. The expression of As-T, an ascidian homolog of the mouse Brachyury (T) gene, starts at the 64-cell stage and is detectable exclusively in the presumptive-notochord blastomeres. The present study shows that presumptive-notochord blastomeres, isolated at the early 32-cell stage, neither differentiates into notochord nor expresses the As-T gene. However, when the presumptive-notochord blastomeres are coisolated or recombined with inducer blastomeres, transcripts of As-T are detected. When presumptive-notochord blastomeres are treated with bFGF, the expression of the As-T gene is also detected. These results suggest that inductive interaction is required for the expression of the As-T gene and that the expression of the As-T gene is closely correlated with the determined state of the notochord-precursor cells (Nakatani, 1996).

To analyse the roles of FGF activity and brachyury (Drosophila homolog: T-related gene) during gastrulation, the consequences of inhibition of FGF-receptor signaling was compared with the phenotype of the zebrafish brachyury homolog mutant, no tail (ntl). Expression of ntl is regulated by FGF and inhibition of FGF receptor-signaling leads to complete loss of the trunk and tail. Since the ntl mutant lacks the tail and notochord but has an otherwise normal trunk, this demonstrates that trunk development is dependent upon an unidentified gene, or set of genes, referred to as no trunk (ntk) which is regulated by FGF. Expression of eve1 and cad1 is also regulated by FGF activity, suggesting that during gastrulation FGF activity is normally restricted to the germ ring where these genes, and ntl, are expressed. Taken together these data suggest that the germ ring acts as a posteriorizing center during AP patterning, mediated by FGF activity in this tissue (Griffin, 1995).

Xenopus FGF receptor-1, which is required for normal development, is stored as a stable, untranslated maternal mRNA transcript in the fully grown immature oocyte, but is translationally activated at meiotic maturation. A short cis-acting element in the FGF receptor 3' untranslated region that inhibits translation of synthetic mRNA. An oocyte cytoplasmic protein specifically binds the 3' inhibitory element, suggesting that translational repression of Xenopus FGF receptor-1 maternal mRNA in the oocyte is mediated by RNA-protein interactions. Such a mechanism of translational control appears to be independent of poly(A) changes (Robbie, 1995).

MAP kinase is involved in mesoderm induction and axial patterning in Xenopus embryos. MAP Kinase Phosphatase (MKP-1) was used to inactivate endogenous MAP kinase. MKP-1 prevents the induction of early and late mesodermal markers by both FGF and activin. In whole embryos, MKP-1 disrupts posterior axial patterning, generating a phenotype similar to that obtained with a dominant inhibitory FGF receptor. Overexpression of either constitutively active MAP kinase or constitutively active MAP kinase (MEK) is sufficient to induce Xbra (Drosophila homology: T-related gene) expression, while only constitutively active MEK iss able to significantly induce expression of muscle actin. When MAP kinase phosphorylation is used as a sensitive marker of FGF receptor activity in vivo, this activity is found to persist at a low and relatively uniform level throughout blastula stage embryos. The finding that a low level of MAP kinase phosphorylation exists in unstimulated animal caps and is absent in caps overexpressing a dominant inhibitory FGF receptor provides a basis for the previous observation that overexpression of this receptor inhibits activin induction. These results indicate that FGF-dependent MAP kinase activity plays a critical role in establishing the responsiveness of embryonic tissues to mesoderm inducers (LaBonne, 1995).

A dorsal-ventral difference in the specification of mesoderm in vivo has been discovered by examining the effect of the dominant-negative FGF receptor on a new member of the Xenopus caudal gene family, Xcad-3. Xcad-3 is expressed throughout the marginal zone during the gastrula stages and serves as a useful marker for events occurring within the mesoderm. Disruption of the FGF signaling pathway by the dominant-negative FGF receptor, disrupts the Xcad-3 expression pattern, eliminating expression preferentially from the dorsal regions of the embryo. The expression of the Xenopus brachyury homolog, Xbra (Drosophila homolog: T-related gene), is more readily eliminated from the dorsal than the ventral region of the embryo by the dominant-negative FGF receptor, indicating that the observed dorsal-ventral differences are not unique to Xcad-3. These results demonstrate the importance of regional effects on FGF-mediated induction in vivo and suggest that FGF-dependent expression of mesodermal genes depends upon the localization of other factors which establish dorsal-ventral differences within the embryo (Northrop, 1994).

Recent studies on Xenopus development have revealed an increasingly complex array of inductive, prepatterning, and competence signals that are necessary for proper mesoderm formation. Fibroblast growth factor (FGF) signals through mitogen-activated protein kinase kinase (MAPKK) to induce mesodermal gene expression. A partially activated form of MAPKK restores expression of the mesodermal genes Xcad-3 and Xbra, eliminated by the dominant-negative FGF receptor (delta FGFR). Expression of a dominant-negative form of MAPKK (MAPKKD) preferentially eliminates the dorsal expression of Xcad-3 and Xbra. Does the regional localization of bone morphogenetic protein-4 (BMP-4) explain why both MAPKKD and delta FGFR eliminate the dorsal but not the ventral expression of Xcad-3 and Xbra? Ectopic expression of BMP-4 is sufficient to maintain the dorsal expression of Xcad-3 and Xbra in embryos containing delta FGFR, and expression of a dominant-negative BMP receptor reduces the dorsal-ventral differences in delta FGFR embryos. These results indicate that regional localization of BMP-4 is responsible for the dorsal-ventral asymmetry in FGF/MAPKK-mediated mesoderm induction (Northrop, 1995).

SH-PTP2, the vertebrate homolog of Drosophila corkscrew, associates with several activated growth factor receptors, but its biological function is unknown. The effects of wild-type and mutant SH-PTP2 RNA were examined on Xenopus embryogenesis. An internal phosphatase domain deletion (delta P) acts as a dominant negative mutant, causing severe posterior truncations. This phenotype is rescued by SH-PTP2, but not by the closely related SH-PTP1. In ectodermal explants, delta P blocks fibroblast growth factor (FGF)- and activin-mediated induction of mesoderm and FGF-induced mitogen-activated protein (MAP) kinase activation. These results indicate that SH-PTP2 is required for early vertebrate development, acting as a positive component in FGF signaling downstream of the FGF receptor and upstream of MAP kinase (Tang, 1995).

Fibroblast growth factor (FGF) signaling has been implicated in the patterning of mesoderm and neural lineages during early vertebrate development. In the mouse, FGF receptor-1 (FGFR1) is expressed in an appropriate spatial and temporal manner to be orchestrating these functions. Fgfr1 is first expressed throughout the primitive ectoderm. At the mid-streak stage, Fgfr1 expression is concentrated in the posterior mesoderm lateral to the primitive streak and is maintained in the migrating mesoderm. Mouse embryos homozygous for a mutated Fgfr1 allele (fgfr1 [delta tmk]) die early in development and show abnormal growth and aberrant mesodermal patterning (Ciruna, 1997).

In Xenopus ectodermal explants (animal caps), fibroblast growth factor (FGF) evokes two major events: induction of ventrolateral mesodermal tissues and elongation. The Xenopus FGF receptor (XFGFR) and certain downstream components of the XFGFR signal transduction pathway (e.g., members of the Ras/Raf/MEK/mitogen-activated protein kinase [MAPK] cascade) are required for both of these processes. Likewise, activated versions of these signaling components induce mesoderm and promote animal cap elongation. Using a dominant negative mutant approach, it has been shown that the protein-tyrosine phosphatase SHP-2 is necessary for FGF-induced MAPK activation, mesoderm induction, and elongation of animal caps. Taking advantage of recent structural information, novel, activated mutants of SHP-2 have been generated. Expression of these mutants induces animal cap elongation to an extent comparable to that evoked by FGF. Surprisingly, however, activated mutant-induced elongation can occur without mesodermal cytodifferentiation and is accompanied by minimal activation of the MAPK pathway and mesodermal marker expression. These results implicate SHP-2 in a pathway(s) directing cell movements in vivo and identify potential downstream components of this pathway. These activated mutants also may be useful for determining the specific functions of SHP-2 in other signaling systems (O'Reilly, 2000).

A chimeric analysis was performed to further study FGFR1 function in the morphogenesis and patterning of the mesodermal germ layer at gastrulation. A population of fgfr1 (delta tmk) embryonic stem cells was established and aggregated with wild-type diploid morulae to generate chimeric embryos. At E9.5, fgfr1(delta tmk)/fgfr1(delta tmk) cells show a marked deficiency in their ability to contribute to the extra-embryonic, cephalic, heart, axial and paraxial mesoderm, and to the endoderm of chimeric embryos. Analyses at earlier stages of development reveal that fgfr1(delta tmk)/fgfr1(delta tmk) cells accumulate within the primitive streak of chimeric embryos, and consequently fail to populate the anterior mesoderm and endodermal lineages at their inception. It is suggested that the primary defect associated with the fgfr1(delta tmk) mutation is a deficiency in the ability of epiblast cells to traverse the primitive streak. Fgfr1 mutant cells are defective in epithelial to mesenchymal transition at the primitive streak stage. fgfr1(delta tmk)/fgfr1(delta tmk) cells that accumulate within the primitive streak of chimeric embryos tend to form secondary neural tubes. These secondary neural tubes are entirely fgfr1(delta tmk)/fgfr1(delta tmk) cell derived. The adoption of ectopic neural fate suggests that normal morphogenetic movement through the streak is essential not only for proper mesodermal patterning but also for correct determination of mesodermal/neurectodermal cell fates. The formation of ectopic neural tissue by mutant cells may demonstrate a neuronal default state for murine embryonic cell (Ciruna, 1997).

Although FGF signaling plays an integral role in the migration and patterning of mesoderm at gastrulation, the mechanism and downstream targets of FGF activity have remained elusive. FGFR1 orchestrates the epithelial to mesenchymal transition and morphogenesis of mesoderm at the primitive streak by controlling Snail and E-cadherin expression. Furthermore, FGFR1 functions in mesoderm cell fate specification by positively regulating Brachyury and Tbx6 expression. Finally, evidence is provided that the attenuation of Wnt3a signaling observed in Fgfr1-/- embryos can be rescued by lowering E-cadherin levels. It is proposed that modulation of cytoplasmic ß-catenin levels, associated with FGF-induced downregulation of E-cadherin, provides a molecular link between FGF and Wnt signaling pathways at the streak (Ciruna, 2001).

Results from the Fgfr1 mutant expression analyses, chimeric studies, and in vitro explant experiments can be assembled into a minimal model for FGFR1 function at gastrulation. This study has defined a specific region of the primitive streak that requires FGFR1 signaling activity; this domain encompasses the paraxial and posterior embryonic mesoderm populations, but excludes the node, axial, and extraembryonic mesoderm. In the context of this domain, it is proposed that FGFR1 signaling orchestrates both the morphogenetic movement and cell fate specification events of gastrulation (Ciruna, 2001).

FGFR1 regulates the morphogenesis and migration of mesodermal cells by differentially regulating intercellular adhesion properties of progenitor populations in the primitive streak. More specifically, FGFR1 signaling is required for the expression of mSnail, a key mediator of epithelial to mesenchymal transitions in development. Furthermore, it is proposed that mSnail expression downstream of FGFR1 is required for the normal downregulation of E-cadherin. Given the morphoregulatory roles for differential cell adhesion during embryogenesis, ectopic E-cadherin expression at the primitive streak of Fgfr1 mutants provides a molecular explanation for the observed defects in epithelial to mesenchymal transition (EMT), progenitor cell migration, and the sorting of Fgfr1-/- from WT cells during gastrulation (Ciruna, 2001).

Beyond its morphoregulatory role at gastrulation, FGFR1 also functions in the specification of mesoderm cell fate. Chimeric analyses demonstrate that FGFR1 is required for T and Tbx6 expression in the primitive streak. The downregulation of T and Tbx6 expression in Fgfr1-/- mesoderm progenitor cells can account for both the reduction of paraxial and posterior mesoderm, and for the formation of ectopic neural tubes observed in Fgfr1 mutant and chimeric analyses. Because studies in zebrafish and Xenopus have also established the function of FGFs in T box gene regulation and posterior mesoderm specification, these results further support an evolutionarily conserved pathway for FGF signaling at gastrulation (Ciruna, 2001).

The mechanisms by which FGFR1 signaling regulates both the morphogenesis and patterning of mesoderm at gastrulation are intricately entwined. Gene dosage and chimeric analyses of Brachyury function have demonstrated that the level of T expression in progenitor cell populations influences the timing and pattern of ingression through the primitive streak. Furthermore, T box genes may also regulate cell adhesion and EMT at gastrulation. In zebrafish, the Brachyury homolog no tail, and the T box gene spadetail have both been implicated as positive regulators of Snail expression. Although regulation of mouse Snail by T has yet to be determined, it is intriguing that in late gastrula-staged Fgfr1 -/- embryos, the only observed domain of mSnail expression overlaps with an Fgfr1-independent domain of T expression at the base of the allantois. Therefore, T may positively regulate Snail expression at the primitive streak, providing another link between Brachyury expression, intercellular adhesion, and the morphogenesis of the mesodermal germ layer (Ciruna, 2001).

In addition, it is proposed that FGFR1 signaling indirectly regulates Wnt signal transduction at the primitive streak. In Fgfr1 -/- embryos, although Wnt3a is expressed in the late primitive streak, direct targets of Wnt signaling (i.e., Brachyury and the T-lacZ reporter transgene) are not activated. It is suggested that ectopic E-cadherin expression in Fgfr1 mutants attenuates Wnt3a signaling by sequestering free ß-catenin from its intracellular signaling pool, and demonstrates that forced downregulation of E-cadherin in Fgfr1 -/- explants can rescue endogenous Wnt signaling at the primitive streak. Evidence that cadherins act as regulators of ß-catenin signaling is well documented. E-Cadherin and LEF-1 bind to partially overlapping sites in the central region of ß-catenin; consequently, LEF-1 and E-cadherin form mutually exclusive complexes with ß-catenin and compete for the same intracellular signaling pool. Furthermore, overexpression of cadherins during Drosophila and Xenopus embryogenesis has been shown to phenocopy Wnt/ß-catenin signaling mutants (Ciruna, 2001).

It is well established that Wnt signaling stabilizes cytosolic levels of ß-catenin by inhibiting its GSK3ß-mediated phosphorylation and degradation. At gastrulation, loss of E-cadherin expression downstream of FGFR1 may also facilitate a rapid intracellular transfer of membrane-bound ß-catenin to the cytosolic 'signaling' pool. Since downregulation of E-cadherin alone is not sufficient to induce ectopic activation of T-lacZ and Brachyury expression in WT primitive streak cultures, signaling through the ß-catenin pathway is still dependent on the activity of localized Wnt signals. However, FGF-mediated changes in cadherin levels and ß-catenin localization could still regulate the threshold for and/or speed of Wnt signaling responses at gastrulation. It is therefore proposed that normal downregulation of E-cadherin at the primitive streak not only regulates the EMT and migration of mesoderm progenitor cells at gastrulation, but also permits the rapid and uninhibited accumulation of cytosolic ß-catenin levels in response to localized Wnt signals. This competition for and opposing influences on the intracellular localization and function of ß-catenin thus establishes a molecular link between the FGF and Wnt signaling pathways at gastrulation. Consequently, FGFR1 activity plays an indirect but permissive role in the propagation of Wnt signaling responses at the primitive streak. The fundamental interregulation of cell adhesion, morphogenesis, and cell fate determination, as demonstrated in this analysis of FGFR1 function, serves to underscore the interdependent nature of morphogenesis and patterning at gastrulation and the intricate network of inductive interactions that pattern and shape the developing embryo (Ciruna, 2001).

Alternative splicing in the fibroblast growth factor receptor 1 (Fgfr1) locus generates a variety of splicing isoforms, including FGFR1alpha isoforms, which contain three immunoglobulin-like loops in the extracellular domain of the receptor. It has been previously shown that embryos carrying targeted disruptions of all major isoforms die during gastrulation, displaying severe growth retardation and defective mesodermal structures. The FGFR1alpha isoforms have been selectively disrupted and they are found to play an essential role in posterior mesoderm formation during gastrulation. The mutant embryos lack caudal somites, develop spina bifida, and die at 9.5-12.5 days of embryonic development because they are unable to establish embryonic circulation. The primary defect is a failure of axial mesoderm cell migration toward the posterior portions of the embryos during gastrulation, as revealed by regional marker analysis and DiI labeling. In contrast, the anterior migration of the notochord is unaffected and the embryonic structures rostral to the forelimb are relatively normal. These data demonstrate that FGF/FGFR1alpha signals are posteriorizing factors that control node regression and posterior embryonic development (Xu, 1999).

Intercellular communication is needed for both the generation of the mesodermal germ layer and its division into distinct subpopulations. To dissect the functions of fibroblast growth factor receptor-1 (FGFR1) during mouse gastrulation as well as to gain insights into its possible roles during later embryonic development, specific mutations have been introduced into the Fgfr1 locus by gene targeting. The results show functional dominance of one of the receptor isoforms and suggest a function for the autophosphorylation of site Y766 in the negative regulation of FGFR1 activity. Y766F and hypomorphic mutations in Fgfr1 generate opposite phenotypes in terms of homeotic vertebral transformations, suggesting a role for FGFR1 in patterning the embryonic anteriorposterior axis by way of regulation of Hox gene activity (Partanen, 1998).

Hypomorphic Fgfr1mutants die neonatally and show posterior truncations, homeotic transformations in the vertebral column (predominantly to the anterior direction), as well as expansion of the limb fields and later distal limb defects. Transformations exclusively to the posterior direction are seen in the gain-of-function Y766F mutants in the absence of other abnormalities. In this respect, the phenotype of Y766F mutants resembles mice carrying loss-of-function mutations in polycomb family members, which are thought to act as negative regulators of Hox complexes. The results are consistent with the data from Xenopus, suggesting a role for FGFs in the positive regulation of Hox gene expression and thus assignment of positional values. The wide spectrum of transformations seen in the Fgfr1 mutants suggests a global role for FGFR1 in the Hox gene regulation. In agreement with this model subtle changes in the early expression patterns of Hox genes, such as HoxD4, HoxB5, and HoxB9, are detected when FGFR1 signal is reduced. These early changes in Hox gene expression and morphological defects suggestive of A-P mispatterning (i.e., limb field expansion) as early as at E8.5 and E9.5, suggest that the function of FGFR1 is in the initial assignment of positional values rather than later in the readout of this information. However, in addition to an early function during gastrulation, FGFR1 may also play a separate role later during vertebral development (Partanen, 1998).

FGF signaling has been implicated in germ layer formation and axial determination. An antibody specific for the activated form of mitogen-activated protein kinase (MAPK) was used to monitor FGF signaling in vivo during early Xenopus development. Activation of MAPK in young embryos is abolished by injection of a dominant negative FGF receptor (XFD) RNA, suggesting that MAPK is activated primarily by FGF in this context. A transition from cytoplasmic to nuclear localization of activated MAPK occurs in morula/blastula stage embryo animal and marginal zones coinciding with the proposed onset of mesodermal competence. It is also possible that the subcellular localization of activated MAPK is part of the actual 'switch' which, once turned on by a putative developmental timer, will allow activated MAPK to activate the FGF signaling pathway as required to respond to mesodermal induction. In Drosophila, a similar phenomenon occurs in the EGFR-dependent and Sevenless-dependent activation of MAPK in which activated MAPK is observed only in the cytoplasm for 2 and 6 hours, respectively, before translocation to the nucleus. These results suggest that an additional regulated step is present in these RTK pathways (Curran, 2000).

Activated MAPK delineates the region of the dorsal marginal zone before blastopore formation and persists in this region during gastrulation, indicating an early role for FGF signaling in dorsal mesoderm. Activated MAPK is also found in posterior neural tissue from late gastrulation onward. Inhibition of FGF signaling does not block posterior neural gene expression (HoxB9) or activation of MAPK; however, inhibition of FGF signaling does cause a statistically significant decrease in the level of activated MAPK. These results point toward the involvement of other receptor tyrosine kinase signaling pathways in posterior neural patterning (Curran, 2000).

The loss of expression of activated MAPK in postinvolution mesoderm may indicate that a specific downregulation of FGF signaling is required for full differentiation of particular mesodermal fates. Activated MAPK expression is lost after the tissue passes over the blastopore lip during involution, though it is maintained in the developing notochord. eFGF and Xbra have similar expressions. Cells overexpressing FGFR1 can not differentiate into myoblasts and FGF signaling can block the differentiation of those cells into muscle. Thus, the downregulation of activated MAPK (and FGF signaling) in more lateral and anterior mesoderm following involution may be necessary for further mesodermal differentiation to proceed. This explanation is also consistent with the evidence for the role of FGF in the maintenance of Xbra expression. A downregulation of FGF signaling following the involution of the anterior mesoderm would eliminate Xbra in that tissue as well. It is not clear how FGF signaling is maintained in some mesoderm and not in others (Curran, 2000).

According to the three-signal model of mesoderm patterning in Xenopus, all mesoderm, with the exception of the Spemann organizer, is originally specified as ventral type, such as lateral plate and primary blood islands. This model proposes that as a result of the antagonistic actions between BMPs and inhibitory factors, mesoderm closest to the Spemann organizer is exposed to the lowest levels of BMPs and is thereby specified as dorsal; conversely, mesoderm farthest away from the Spemann organizer is exposed to the highest levels of BMPs and is specified as ventral. In this model, a gradient of BMP activity is generated in the marginal zone through the action of the Spemann organizer. The Spemann organizer is the source of a number of secreted factors, including noggin, chordin, follistatin, and Xnr-3, that antagonize the activity of a uniformly expressed field of BMPs in the marginal zone. This model would appear to give a molecular explanation for the observed activities of the Spemann organizer and is consistent with predictions of the widely accepted three-signal model of mesoderm patterning (Kumano, 2000 and references therein).

Thus it has been proposed that the blood islands become restricted to the ventralmost mesoderm because they are not exposed to the BMP-inhibiting activity of the Spemann organizer. Evidence is presented here that, contrary to predictions of this model, the blood islands remain ventrally restricted even in the absence of Spemann organizer signaling. Inhibition of FGF signaling with a dominant negative receptor results in the expansion of the blood island-forming territory with a concomitant loss of somite. The requirement for FGF signaling in specifying somite versus blood island territories is observed as early as midgastrulation. The nonoverlapping expression domains of Xnr-2 and Xbra in the gastrula marginal zone appear to mark presumptive blood island and somite, respectively. Inhibition of FGF signaling with dominant negative receptor leads to an expansion of Xnr-2 expression and to a corresponding reduction in Xbra expression. However, no evidence is found that manipulation of BMP signaling, either positively or negatively, alters the expression domains of Xnr-2 and Xbra. These results suggest that FGF signaling, rather than BMP-inhibiting activity, is essential for restriction of the ventral blood islands to ventral mesoderm (Kumano, 2000).

Xenopus laevis embryogenesis is controlled by the inducing activities of Spemann's organizer. These inducing activities are separated into two distinct suborganizers: a trunk organizer and a head organizer. The trunk organizer induces the formation of posterior structures by emitting signals and directing morphogenesis. The fibroblast growth factor receptor (FGFR) signaling pathway, also known to regulate posterior development, performs critical functions within the cells of Spemann's organizer. Specifically, the FGFR pathway is required in the organizer cells in order for those cells to induce the formation of somitic muscle and the pronephros. The organizer influences the differentiation of somitic muscle and the pronephros by emitting signals that pattern the mesodermal germ layer; FGFR regulates the production of these signals. In addition, the FGFR pathway is required for the expression of chordin, an organizer-specific protein required for the trunk-inducing activities of Spemann's organizer. Significantly, the FGFR pathway has a minimal effect on the function of the head organizer. The FGFR pathway is a defining molecular component that distinguishes the trunk organizer from the head organizer by controlling the expression of organizer-specific genes required to induce the formation of posterior structures and somitic muscle in neighboring cells (Mitchell, 2001).

Thus, normal embryonic development results from a balance between antagonistic mechanisms that affect cell fate. An important role of the FGFR pathway is to control the activities of Spemann's organizer that are required for the development of the trunk and somitic muscle. The FGFR pathway may exert its effects on organizer function by regulating mechanisms that attenuate BMP signaling. One obvious mechanism by which the FGFR pathway may antagonize BMP signaling is by controlling the expression of proteins that function as BMP antagonists. Expression of the chordin protein, a protein that antagonizes BMP signaling and is necessary for the trunk/tail-inducing functions of Spemann's organizer, requires the FGFR pathway. In addition, the FGFR pathway may also inhibit BMP signaling by a more direct mechanism. There are several examples that demonstrate that activation of tyrosine kinase receptors, such as the FGF receptor, can inhibit BMP signal transduction. For instance, the Smad1 transcription factor is activated in response to BMP signaling, but Smad1 function is inhibited by activation of the MAPK proteins, known signaling components of the FGFR pathway. Thus, the FGFR and BMP signaling pathways can compete directly for alternate modes of regulation of common downstream signaling components. Therefore, a critical role of the FGFR pathway in Spemann's organizer may be to attenuate BMP signaling both indirectly by controlling chordin gene expression and directly by regulating common downstream signaling components such as Smad1 (Mitchell, 2001).

A number of studies indicate that the FGFR pathway and signaling centers act in concert to control various aspects of vertebrate development. Limb formation is governed by the activity of the AER signaling center, while tooth development is controlled by the activities of the primary enamel knot signaling center. Significantly, both limb formation and tooth development also require a functional FGFR pathway, but it has been difficult to assess directly whether the FGFR pathway is actually required within these signaling centers themselves. However, these studies do provide evidence that the FGFR pathway antagonizes BMP signaling associated with these signaling centers. Spemann's organizer, an evolutionary conserved and fundamental signaling center that controls the formation of the vertebrate body plan and associated tissues, requires the FGFR pathway for normal function. These results, combined with observations associated with other signaling centers, lead to the proposal that a balance between the FGFR and BMP signaling pathways is a fundamental mechanistic feature controlling the formation and function of all vertebrate signaling centers. Given the evolutionary conservation and relevance of the FGFR pathway to vertebrate signaling centers in general, a rigorous dissection of the FGFR pathway's role in Spemann's organizer should continue to provide insights into the fundamental mechanisms that regulate vertebrate development (Mitchell, 2001).

Anteroposterior (AP) patterning of the developing neural tube is crucial for both regional specification and the timing of neurogenesis. Several important factors are involved in AP patterning, including members of the WNT and FGF growth factor families, retinoic acid receptors, and HOX genes. The interactions between FGF and retinoic signaling pathways have been studied. Blockade of FGF signaling downregulates the expression of members of the RAR signaling pathway, RARalpha, RALDH2 and CYP26. Overexpression of a constitutively active RARalpha2 rescues the effects of FGF blockade on the expression of XCAD3 and HOXB9. This suggests that RARalpha2 is required as a downstream target of FGF signaling for the posterior expression of XCAD3 and HOXB9. Surprisingly, it was found that posterior expression of FGFR1 and FGFR4 is dependent on the expression of RARalpha2. Anterior expression is also altered with FGFR1 expression being lost, whereas FGFR4 expression is expanded beyond its normal expression domain. RARalpha2 is required for the expression of XCAD3 and HOXB9, and for the ability of XCAD3 to induce HOXB9 expression. It is concluded that RARalpha2 is required at multiple points in the posteriorization pathway, suggesting that correct AP neural patterning depends on a series of mutually interactive feedback loops among FGFs, RARs and HOX genes (Shiotsugu, 2004).

FGF receptor and mesenchymal-epithelial signaling

Four FGF receptor genes have been identified in mammals (Fgfr1 to Fgfr4), each comprising an extracellular domain composed of two or three immunoglobulin-like (Ig) loops, a transmembrane segment and an intracellular tyrosine kinase. The fibroblast growth factor receptor 2 gene is differentially spliced to encode two transmembrane tyrosine kinase receptor proteins that have different ligand-binding specificities and exclusive tissue distributions. Ligand-binding specificity for FGF receptors is mediated by the second and third Ig-loops. For Fgfr1-Fgfr3, the third Ig loop is encoded by two exons, an invariant exon termed IIIa and one of two exons, termed IIIb and IIIc, respectively, to which the IIIa exon is spliced. This generates two receptor isoforms with quite different ligand-binding specificities. Fgfr2 containing the IIIb exon is found mainly in epithelia, and is activated by four known ligands (Fgf1, Fgf3, Fgf7 and Fgf10), which are synthesized predominantly in the tissue mesenchyme. In contrast, Fgfr2(IIIc) is located primarily in the mesenchyme, and apart from Fgf1, which binds to all known receptors, Fgfr2(IIIc) is activated by a different set of Fgf ligands. Hence, Fgfr2(IIIb) and Fgfr2(IIIc) are expressed in mutually exclusive cell lineages, using a positively regulated splicing mechanism that involves intron sequences adjacent to the isoform-specific exons. Cre-mediated excision was used to generate mice lacking the IIIb form of fibroblast growth factor receptor 2 while retaining expression of the IIIc form. Fibroblast growth factor receptor 2(IIIb) null mice are viable until birth, but have severe defects of the limbs, lung and anterior pituitary gland. The development of these structures appears to initiate, but then fails with the tissues undergoing extensive apoptosis. There are also developmental abnormalities of the salivary glands, inner ear, teeth and skin, as well as minor defects in skull formation. These findings point to a key role for fibroblast growth factor receptor 2(IIIb) in mesenchymal-epithelial signaling during early organogenesis (De Moerlooze, 2000).

FGFR and regeneration in planarians

The study of planarian regeneration may help gain an understanding of how organs and tissues can be rebuilt after injury, disease or ageing. The robust regenerative abilities of planarians are based upon a population of totipotent stem cells (neoblasts), and among the organs regenerated by these animals is a well-organized central nervous system. In recent years, methodologies such as whole-mount in situ hybridizations and double-stranded RNA have been extended to planarians with the aim of unravelling the molecular basis of their regenerative capacities. nou-darake (ndk), a gene encoding a fibroblast growth factor receptor (FGFR)-like molecule is specifically expressed in the head region of the planarian Dugesia japonica. Loss of function of ndk by RNA interference results in the induction of ectopic brain tissues throughout the body. This ectopic brain formation is suppressed by inhibition of two planarian FGFR homologs (FGFR1 and FGFR2). Additionally, ndk inhibits FGF signaling in Xenopus embryos. The data suggest that ndk may modulate FGF signaling in stem cells to restrict brain tissues to the head region of planarians (Cebria, 2002).

How could the silencing of a gene specifically expressed in the planarian head lead to the differentiation of brain-like tissues throughout the body in a non-autonomous cell manner? Sequence analyses, in situ hybridization data, RNAi experiments and mRNA injections in Xenopus show that NDK has potential FGF binding domains, is expressed in the head of planarians, restricts brain differentiation to the planarian head region, and is capable of inhibiting FGF signaling. Then, one simple model would be that NDK may restrict the diffusion range from a putative source of brain-inducing factors (FGF or FGF-like molecules) in the planarian head to the rest of the body. Loss of function of ndk, therefore, would allow these factors to diffuse to posterior regions, and interact with FGF receptors outside of the head region, thus triggering ectopic brain formation. The observation of a gradual brain expansion to more posterior regions in dsRNA-injected animals supports this idea. Since these hypothetical brain-inducing factors must diffuse distances of several millimetres between the planarian head and the posterior regions where the ectopic brain is formed, diffusion rates as well as the role of extracellular matrix components during this process should be analysed (Cebria, 2002).

Even though FGF-like ligands in planarians have yet to be identifed, ndk provides strong molecular evidence for the existence of a brain-inducing circuit based on the FGF signaling pathway. Whereas antagonists of BMP4 are believed to be the main neural inducers in vertebrates, recent work has suggested that FGFs are important in neural formation and patterning. For instance, BMP antagonists do not induce neural tissues in the presence of dominant-negative FGF receptors in Xenopus. In addition, studies in chicken have shown that neural induction by BMP antagonists requires FGF signaling, suggesting that FGFs, as in planarians, may be essential for neural tissue formation in the vertebrates. The fact that planarian ndk can functionally inhibit Xenopus bra activation during Xenopus gastrulation raises the possibility that the vertebrate homolog of ndk may be modulating FGF signaling. Further studies will be required to understand the extent of the involvement of ndk in vertebrate organogenesis -- in particular, neurogenesis (Cebria, 2002).

Table of contents

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

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