FGF receptor 1
Genes coding for FGF receptors Fibroblast growth factors (FGFs) mediate
many cell-cell signaling events during early development.
While the actions of FGFs have been well-studied, the
roles played by specific members of the FGF receptor
(FGFR) family are poorly understood. To characterize the
roles played by individual FGFRs, the regulation
and expression of the three Xenopus FGFRs described
to date (XFGFR-1, XFGFR-2, and XFGFR-4) have been characterized.
(1) The expression of Xenopus FGFR-4 is present as a maternal mRNA and is found in
the embryo through at least the tadpole stage. XFGFR-4
and XFGFR-1 mRNAs are present at comparable levels,
which argues that both mediate FGF signaling during early development.
(2) In animal
caps, the expression of XFGFR-4 differs from the expression of XFGFR-1 and
XFGFR-2, suggesting that the FGFRs are independently
regulated in ectoderm. (3) Using whole-mount in situ
hybridization, it has been shown that XFGFR-1, XFGFR-2, and
XFGFR-4 are expressed in dramatically different patterns,
arguing that specific FGF signaling events are mediated
by different members of the FGFR family. Among
these, FGF signaling during the induction of neural crest
cells is likely to be mediated by XFGFR-4. Comparison
of these results with previously reported FGFR expression
patterns reveals that FGFR-1 expression is highly conserved
among vertebrate embryos, and FGFR-2 expression
shows many features that are conserved and some
that are divergent. The distinct patterns of expression of FGFR-4
mRNAs in different vertebrate embryos, together with
the relatively high divergence of FGFR-4 sequences
(compared to the other FGFRs), argues that FGFR-4
genes may play different roles in development in different
taxa. In particular, in frogs and fish, FGFR-4 may act
in signaling between rhombomeres, and in induction of
the neural crest. Because FGFR-4 expression is different
in mice and chicks, FGFR-4 is unlikely to serve similar
roles in these vertebrates (Golub, 2000).
There are four mammalian fibroblast growth factor receptors, FGFRs 1-4, three of which (FGFRs1-3) are known to play important roles in skeletal differentiation and growth, as revealed by the identification of specific mutations in previously recognized clinical syndromes. These proteins are transmembrane receptors with three immunoglobulin-like domains (IgI, IgII and IgIII) in the extracellular region and a split tyrosine kinase domain in the intracellular region of the molecule. They all show high affinity for fibroblast growth factors (FGFs), of which fourteen mammalian forms are known. The ligand binding region is thought to include parts of IgII and IgIII domains, but the precise region of binding is FGF-specific. FGFRs1-3 each exist as two principal isoforms, derived from alternative splicing of a common IgIIIa exon onto either a IIIb or a IIIc exon. For FGFR2, the variant including the IIIa/IIIb domain is also known as KGFR, and the variant including the IIIa/IIIc domain is known as BEK. Both isoforms bind with high affinity to FGF1 and FGF4, but differ in their affinity for FGF2 (high affinity for IgIIIa/IgIIIc domains) and FGF7 (high affinity for IgIIIa/IgIIIb domains). These binding properties are reflected in the mitogenic activity of each FGF in cell lines expressing specific FGFR splice variants (Iseki, 1997 and references).
The proper guidance of the C. elegans hermaphrodite sex myoblasts (SMs) requires the genes egl-15 and egl-17. egl-15 has been shown to encode the C. elegans orthologue of the fibroblast growth factor receptor. egl-17 was cloned and is a member of the fibroblast growth factor family, one of the first functional invertebrate FGFs known. egl-17 shares homology with other FGF members, conserving the key residues required to form the distinctive tertiary structure common to FGFs. The SM migration defect seen in egl-17 mutant animals represents complete loss of egl-17 function. While mutations in egl-17 affect only SM migrations, mutations in egl-15 can result in larval arrest, and scrawny body morphology (Burdine, 1997).
Receptor tyrosine phosphatases have been implicated in playing important roles in cell signaling events due to their ability to regulate the level of protein tyrosine phosphorylation. Although the catalytic activity of their phosphatase domains have been well established, the biological roles of these molecules are, for the most part, not well understood. The Caenorhabditis elegans protein CLR-1 (CLeaR) is a receptor tyrosine phosphatase (RTP) with a complex extracellular region and two intracellular phosphatase domains. Mutations in clr-1 result in a dramatic Clr phenotype that has been used to study the physiological requirements for the CLR-1 RTP. Animals homozygous for the clr-1(e1745ts) mutation appear completely wild-type at the permissive temperature of 15°C but display a severe Clr phenotype when raised at the nonpermissive temperature of 25°C. Clr animals are extremely short, immobile, and infertile, and their pharynx and intestine appear to float within the pseudocoelom. The cells and cell processes in these animals appear to be separated from each other, a phenotype that has been used extensively to visualize neuronal processes and cell boundaries in C. elegans. These abnormalities all appear to result from clear fluid accumulating in the pseudocoelom. Whereas some animals homozygous for the most severe alleles of clr-1 die during larval development with hypodermal ruptures, most remain alive and display an extreme Clr phenotype. The biological basis of the Clr phenotype is not understood, although it mimics the phenotype observed when cells in the C. elegans excretory system or the CAN neurons are destroyed by laser microsurgery. The complete suppressibility of the Clr phenotype by mutations in egl-15 does not necessarily indicate that CLR-1 acts only on a component of the FGFR signaling pathway. It is possible that CLR-1 has roles in other pathways as well, but that its failure to function in these pathways does not result in an obvious phenotypic effect in a wild-type background. In fact, mutations in clr-1 can partially suppress the effect of mutations that compromise the vulval induction pathway mediated by the epidermal growth factor receptor (EGFR) homolog LET-23. Thus, CLR-1 may partially antagonize signaling mediated by both FGFR and EGFR tyrosine kinases. Such other roles of CLR-1 might not be revealed by clr-1 mutations in a wild-type background if the other pathways affected by CLR-1 have different primary negative regulatory elements (Kokel, 1998).
Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson-Weiss,
and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast
growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that
potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine
mutations, the mechanism of receptor activation remains unclear. The effect of two of
these mutations, W290G and T341P, was examined on receptor dimerization and activation. These mutations result
in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in
full-length FGFR2, the mutations induce receptor dimerization and elevated levels of tyrosine kinase
activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase
activity are all abolished if either the W290G or the T341P mutation is expressed in conjunction
with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These
results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by
noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate
FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the
formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in
intermolecular disulfide bonding, resulting in constitutive activation of the receptor (Robertson, 1998).
Activation of FGF receptors necessitates ligand induced dimerization. Dimerization of FGF receptors is mediated by either soluble or cell surface-bound heparin sulfate proteoglycans, which in concert with FGFs promote receptor dimerization, activation, and induction of biological responses. The cytoplasmic domain of mammalian FGFR1 contains at least seven tyrosine autophosphorylation sites. Autophosphorylation of two tyrosine residues in the catalytic domain is critical for the kinase activity of FGFR1. Another C-terminal tail tyrosine functions as a binding site for phospholipase C-gamma, and is essential for FGF-induced stimulation of phosphatidylinositol hydrolysis. Analysis of the crystal structure of the tyrosine kinase domain reveals that residues in the activation loop appear to interfere with substrate peptide binding ("substrate" refers to the downstream target of FGF-R signaling), but not with ATP binding, revealing a general autoinhibitory mechanism for receptor tyrosine kinases (Mohammadi, 1996 and references).
A cDNA clone isolated from a Xenopus embryo (stage 8 blastula) library, was predicted to encode a variant form of the type 1 fibroblast growth factor receptor
(FGFR1) containing a dipeptide Val-Thr (VT) deletion at amino acid positions 423 and 424 located
within the juxtamembrane region.
Sequence analysis of genomic DNA encoding a portion of the FGFR1 juxtamembrane region
demonstrates that this variant form arises from use of an alternative 5' splice donor site. RNase
protection analysis revealed that both VT- and VT+ forms of the FGFR1 are expressed throughout
embryonic development, VT+ being the major form. Amino acid position 424 is located within a
consensus sequence for phosphorylation by a number of Ser/Thr kinases. A VT+
peptide is specifically phosphorylated by protein kinase C (See Drosophila PKC) in vitro, but not by protein kinase A
(PKA).
In contrast, the VT- peptide is not a substrate for either enzyme. Phosphorylation
levels of in vitro synthesized FGFR-VT+ protein by PKC are twice that of FGFR-VT- protein. In a
functional assay, Xenopus oocytes expressing FGFR-VT- or FGFR-VT+ protein are equally able to
mobilize intracellular Ca2+ in response to basic fibroblast growth factor (bFGF). However,
pretreatment with phorbol ester significantly reduces this mobilization in oocytes
expressing FGFR-VT+ while having little effect on oocytes expressing FGFR-VT-. These findings
demonstrate that alternative splicing of Val423-Thr424 generates isoforms that differ in their ability
to be regulated by phosphorylation; thus, alternative splicing represents an important mechanism for regulating FGFR
activity (Gillespie, 1995).
Fibroblast growth factor 1 (FGF-1) induces neurite outgrowth in PC12 cells. The FGF receptor
1 (FGFR-1) has been shown to be much more potent than FGFR-3 in the induction of neurite outgrowth. Advantage was taken of this difference to identify the cytoplasmic regions of
FGFR-1 that are responsible for the induction of neurite outgrowth in PC12 cells; receptor chimeras were prepared containing different regions of the FGFR-1 introduced into the FGFR-3 protein. The chimeric
receptors were introduced into FGF-nonresponsive variant PC12 cells (fnr-PC12 cells), and their ability to mediate
FGF-stimulated neurite outgrowth of the cells was assessed. The juxtamembrane (JM) and carboxy-terminal (COOH) regions
of FGFR-1 were identified as conferring robust and moderate abilities, respectively, for induction of neurite outgrowth to
FGFR-3. Analysis of FGF-stimulated activation of signal transduction reveals that the JM region of FGFR-1 confers
strong and sustained tyrosine phosphorylation of several cellular proteins and activation of MAP kinase. The SNT/FRS2
protein was demonstrated to be one of the cellular substrates preferentially phosphorylated by chimeras containing the JM
domain of FGFR-1. SNT/FRS2 links FGF signaling to the MAP kinase pathway. Thus, the ability of FGFR-1 JM domain
chimeras to induce strong sustained phosphorylation of this protein would explain the ability of these chimeras to activate MAP
kinase and hence neurite outgrowth. The role of the COOH region of FGFR-1 in induction of neurite outgrowth involves the
tyrosine residue at amino acid position 764, a site required for phospholipase C gamma binding and activation, whereas the JM
region functions primarily through a non-phosphotyrosine-dependent mechanism. In contrast, assessment of the chimeras in
the pre-B lymphoid cell line BaF3 for FGF-1-induced mitogenesis reveals that the JM region does not play a role in this cell
type. These data indicate that FGFR signaling can be regulated at the level of intracellular interactions and that signaling
pathways for neurite outgrowth and mitogenesis use different regions of the FGFR (Lin, 1998).
Binding of FGF receptor to FGF The fibroblast growth factor (FGF) family plays key roles in development, wound healing, and angiogenesis. An understanding of the molecular nature of the
interactions of FGFs with their receptors (FGFRs) has been seriously limited by the absence of structural information on FGFR or FGF-FGFR complex. In
this study, based on an exhaustive analysis of the primary sequences of the FGF family, it has been determined that the residues that constitute the primary
receptor-binding site of FGF-2 are conserved throughout the FGF family, whereas those of the secondary receptor binding site of FGF-2 are not. It is
proposed that the FGF-FGFR interaction mediated by the 'conserved' primary site interactions is likely to be similar if not identical for the entire FGF family,
whereas the 'variable' secondary sites, on both FGF as well as FGFR mediate specificity of a given FGF to a given FGFR isoform. Furthermore, since the
pro-inflammatory cytokine interleukin 1 (IL-1) and FGF-2 share the same structural scaffold, the spatial orientation of the primary
receptor-binding site of FGF-2 is found to coincide structurally with the IL-1beta receptor-binding site when the two molecules are superimposed. The structural
similarities between the IL-1 and the FGF system provided a framework to elucidate molecular principles of FGF-FGFR interactions. In the FGF-FGFR
model proposed here, the two domains of a single FGFR wrap around a single FGF-2 molecule such that one domain of FGFR binds to the primary
receptor-binding site of the FGF molecule, while the second domain of the same FGFR binds to the secondary receptor-binding site of the same FGF
molecule. Finally, the proposed model is able to accommodate not only heparin-like glycosaminoglycan (HLGAG) interactions with FGF and FGFR but
also FGF dimerization or oligomerization mediated by HLGAG (Venkataraman, 1999).
To elucidate the structural determinants governing specificity in fibroblast growth factor (FGF) signaling, the crystal structures were determined for FGF1 and
FGF2 complexed with the ligand binding domains (immunoglobulin-like domains 2 [D2] and 3 [D3]) of FGF receptor 1 (FGFR1) and FGFR2, respectively. Highly
conserved FGF-D2 and FGF-linker (between D2-D3) interfaces define a general binding site for all FGF-FGFR complexes. Specificity is achieved through
interactions between the N-terminal and central regions of FGFs and two loop regions in D3 that are subject to alternative splicing. These structures provide a
molecular basis for FGF1 as a universal FGFR ligand and for modulation of FGF-FGFR specificity through primary sequence variations and alternative splicing (Plotnikov, 2000).
Phosphorylation and signaling downstream of FGF receptor p70(s6k) has a role in cell cycle progression in response to specific extracellular stimuli. The signal transduction pathway
leading to activation of p70(s6k) by fibroblast growth factor receptor-1 (FGFR-1) was examined in FGF-2-treated rat L6
myoblasts. p70(s6k) is activated in a biphasic and rapamycin-sensitive manner. Although phosphatidylinositol 3'-kinase is
not activated in the FGF-2 treated cells, as judged from in vitro and in vivo analyses, wortmannin and LY294002 treatment
inhibits p70(s6k) activation. The involvement of S6 kinase in FGFR-1-dependent biological responses was examined in murine
brain endothelial cells. In response to FGF-2, these cells differentiate to form tube-like structures in collagen gel cultures and
proliferate when cultured on fibronectin. p70(s6k) is not activated in endothelial cells on collagen, whereas activation is
observed during proliferation on fibronectin. These results indicate that FGFR-1 mediates p70(s6k) activation by a phosphatidylinositol
3'-kinase-independent mechanism that does not require PKC activation; furthermore, proliferation, but not differentiation
of endothelial cells in response to FGF-2, is associated with p70(s6k) activation (Kanda, 1997).
Fibroblast growth factor receptor 3 (FGFR3) mutations are frequently involved in human developmental disorders and cancer. Activation of FGFR3, through mutation or ligand stimulation, results in autophosphorylation of multiple tyrosine residues within the intracellular domain. To assess the importance of the six conserved tyrosine residues within the intracellular domain of FGFR3 for
signaling, derivatives were constructed containing an N-terminal myristylation signal for plasma membrane localization and a point mutation (K650E) that confers constitutive kinase activation. A derivative containing all conserved tyrosine residues stimulates cellular transformation and activation of several FGFR3 signaling pathways. Substitution of all nonactivation loop tyrosine residues with phenylalanine renders this FGFR3 construct inactive, despite the presence of the activating K650E mutation. Addition of a single tyrosine residue, Y724, restores its ability to stimulate cellular transformation, phosphatidylinositol 3-kinase activation, and phosphorylation of Shp2, MAPK, Stat1, and Stat3. These results demonstrate a critical role for Y724 in the activation of multiple signaling pathways by constitutively activated mutants of FGFR3 (Hart, 2001).
In looking for novel factors involved in the regulation of the fibroblast growth factor (FGF) signaling pathway, a zebrafish sprouty4 gene was isolated, based on its extensive similarities with the expression patterns of both fgf8
and fgf3. Through gain- and loss-of-function experiments, it has been demonstrated that Fgf8 and Fgf3 act in vivo to induce the
expression of Spry4, which in turn can inhibit activity of these growth factors. When overexpressed at low doses,
Spry4 induces loss of cerebellum and reduction in size of the otic vesicle, thereby mimicking the fgf8/acerebellar
mutant phenotype. Injections of high doses of Spry4 cause ventralization of the embryo, a phenotype opposite that of the dorsalization induced by
overexpression of Fgf8 or Fgf3. Conversely, inhibition of Spry4 function through injection of antisense morpholino oligonucleotide
leads to a weak dorsalization of the embryo, the phenotype expected for an upregulation of Fgf8 or Fgf3 signaling pathway. Finally, it has been shown that
Spry4 interferes with FGF signaling downstream of the FGF receptor 1 (FGFR1). In addition, this analysis reveals that signaling through
FGFR1/Ras/mitogen-activated protein kinase pathway is involved, not in mesoderm induction, but in the control of the dorsoventral patterning via the
regulation of bone morphogenetic protein (BMP) expression (Furthauer, 2001).
The zebrafish Spry cDNA codes for a 310 amino acid protein. It is most closely related to mouse Sprouty4, the two proteins displaying 65.7% overall amino acid
similarity while showing less than 50% amino acid similarity with the mouse or human Spry1, Spry2 and Spry3. Phylogenetic analysis further
confirms that this clone encodes a zebrafish Sprouty4 homolog. Alignment of the peptide sequence of the sprouty genes reveals the existence of three
domains of particularly extensive conservation. Most prominent among these is the C-terminal 130 amino acid cysteine-rich domain, which constitutes the
distinctive feature of Spry proteins and has been shown to be sufficient for the localization of Spry at the plasma membrane. In zebrafish Spry4
this domain contains 25 cysteine residues, 17 of which are found at conserved positions in all Spry proteins (Furthauer, 2001).
To investigate at which level Spry4 interferes with FGF signaling, an assessment was made of its ability to rescue a constitutively active (CA) FGFR1-induced dorsalization. Coinjection of CA-FGFR1 with increasing doses of spry4 mRNA progressively rescues this dorsalization phenotype. For 125 pg spry4 mRNA, only 29% (32/109) embryos remain dorsalized while using 250 pg led to a complete rescue of the dorsalization phenotype. This clearly demonstrates that spry4 antagonizes the FGF signaling mediated through FGFR1. Stimulation of FGFR1 ultimately leads to the phosphorylation of the extracellular-regulated protein kinases (ERK) 1 and 2. Therefore advantage was taken of the use of an antibody recognizing the activated form of ERK to estimate the effect of Spry4 on MAPK activity. In
accordance with an activation of ERK after the stimulation of FGFR1, localized misexpression of CA-FGFR1 induces ectopic activation of MAPK at blastula stage,
whereas activated MAPK is barely detectable in wild-type control embryos. Conversely, localized injection of 250 pg spry4 mRNA causes a
local inhibition of MAPK activation at mid-gastrula stages, when the MAPK is ubiquitously activated in wild-type embryos. These results therefore demonstrate that spry4 interferes with FGF signaling by acting downstream of FGFR1, leading to a subsequent downregulation of MAPK activity (Furthauer, 2001).
FGFs mediate their pleiotropic responses by binding to and activating a family of receptor tyrosine kinases (RTKs) designated FGF receptors (FGFR) 1-4. Many of the cellular responses of FGFs are mediated by the membrane-linked docking proteins, FRS2alpha and FRS2beta, that have no closely related Drosophila homologs. Both FRS2alpha and FRS2beta contain myristyl anchors and phosphotyrosine binding (PTB) domains in their N termini and multiple tyrosine phosphorylation sites in their C termini that serve as binding sites for the adaptor protein Grb2 and the protein tyrosine phosphatase Shp2. The docking protein FRS2alpha functions as a major mediator of signaling by FGF and NGF receptors. In addition to tyrosine phosphorylation, FRS2alpha is phosphorylated by MAP kinase on multiple threonine residues in response to FGF stimulation or by insulin, EGF, and PDGF, extracellular stimuli that do not induce tyrosine phosphorylation of FRS2alpha. Prevention of FRS2alpha threonine phosphorylation results in constitutive tyrosine phosphorylation of FRS2alpha in unstimulated cells and enhanced tyrosine phosphorylation of FRS2alpha, MAPK stimulation, cell migration, and proliferation in FGF-stimulated cells. Expression of an FRS2alpha mutant deficient in MAPK phosphorylation sites induces anchorage-independent cell growth and colony formation in soft agar. These experiments reveal a novel MAPK-mediated, negative feedback mechanism for control of signaling pathways that are dependent on FRS2alpha and a mechanism for heterologous control of signaling via FGF receptors (Lax, 2002).
Convergent extension behavior is critical for the formation of the vertebrate body axis. In Xenopus, components of the Wnt signaling pathway have been shown to be required for convergent extension movements but the relationship between cell fate and morphogenesis is little understood. Loss of function analysis indicates that Xnr3 activates Xbra expression through FGFR1. It is shown that eFGF activity is not essential in the pathway, and that dishevelled acts downstream of Xnr3 and not in a parallel pathway. Evidence is presented for the involvement of the EGF-CFC protein FRL1, and it is suggested that the pro-domain of Xnr3 may be required for its activity. Since Xnr3 is a direct target of the maternal ßcatenin/XTcf3 signaling pathway, Xnr3 provides the link between the initial, maternally controlled, allocation of cell fate, and the morphogenetic movements of cells derived from the organizer (Yokota, 2003).
How does Xnr3 activate the FGF receptor? The activity of an intermediary
such as eFGF seems unlikely since depletion of eFGF with a morpholino oligo has no effect on Xbra expression or explant elongation. Another interesting possibility is that Xnr3 activation of FGFR depends upon an EGF-CFC protein that was first isolated as an FGFR binding protein, FRL1. Over-expression of FRL1 mRNA causes elongation and NCAM and MyoD expression in animal caps, as well as the formation of finger-like protrusions in whole embryos. Xnr3 and FRL1 synergize strongly in animal cap assays. Direct tests
of FRL1 function and of its possible interactions with nodal proteins are required to determine its role (Yokota, 2003).
Fibroblast growth factors (FGFs) are pleiotrophic growth factors that control cell proliferation, migration, differentiation and embryonic patterning. During early zebrafish embryonic development, FGFs regulate dorsoventral patterning by controlling ventral bone morphogenetic protein (BMP) expression. FGFs function by binding and activating high-affinity tyrosine kinase receptors. FGF activity is negatively regulated by members of the Sprouty family, which antagonize Ras signalling induced by receptor tyrosine kinases. On the basis of similarities in their expression patterns during embryonic development, five genes have been identified that define a synexpression group -- fgf8, fgf3, sprouty2, sprouty4, as well as a novel gene, sef (similar expression to fgf genes). Sef encodes a conserved putative transmembrane protein that shares sequence similarities with the intracellular domain of the interleukin 17 receptor. In zebrafish, Sef functions as a feedback-induced antagonist of Ras/Raf/MEK/MAPK-mediated FGF signalling (Fürthauer, 2002).
Twist is a potential regulator of FGF receptor Mesodermal development is a multistep process in which cells become increasingly specialized to form
specific tissue types. In Drosophila and mammals, proper segregation and patterning of the mesoderm
involves the bHLH factor Twist. The activity of a Twist-related factor, CeTwist, was investigated during
Caenorhabditis elegans mesoderm development. Within the bHLH domain, CeTwist shares
59%-63% identity to published Twist family members in other species. Outside of the bHLH domain, there is no obvious
homology between CeTwist and other Twist family members. Embryonic mesoderm in C. elegans derives from a number
of distinct founder cells that are specified during the early lineages; in contrast, a single blast cell (M) is
responsible for all nongonadal mesoderm formation during postembryonic development. Using
immunofluorescence and reporter fusions, the activity pattern of the gene encoding CeTwist was determined.
No activity is observed during specification of mesodermal lineages in the early embryo; instead, the gene
is active within the M lineage and in a number of mesodermal cells with nonstriated muscle fates (Harfe, 1998).
A role
for CeTwist in postembryonic mesodermal cell fate specification was indicated by ectopic expression and
genetic interference assays. These experiments show that CeTwist is responsible for activating two
target genes normally expressed in specific subsets of nonstriated muscles derived from the M lineage. In
vitro and in vivo assays suggest that CeTwist cooperates with the C. elegans E/Daughterless homolog in
directly activating these targets. The two target genes that have been studied, ceh-24 and egl-15, encode an
NK-2 class homeodomain (Drosophila homologs Tinman and Bagpipe) and an FGF receptor (FGFR) homolog, respectively. egl-15 encodes a member of the FGF receptor (FGFR) family that is required for the proper migration of the sex myoblasts. The egl-15 promoter is active in many early M lineage descendants and later
in the four vm1 vulval muscles. Although this promoter activity pattern is distinct from
that of ceh-24, the activity of each in the later M lineage suggested the possibility of a common factor specifying M lineage
activity for the two genes. An egl-15 promoter fragment of 701 bp is sufficient to drive reporter expression in the M lineage.
This fragment contained five matches to the E-box consensus. Three were precise NdE boxes (CATATG), whereas the
other two differed from this consensus by a single base pair. Deletion analysis suggests critical roles for the
E-box motif and for additional elements in egl-15 promoter activity: specifically, M-lineage activity is eliminated by
promoter truncations that remove the first two NdE-like boxes, and by a deletion that
removes the three proximal NdE-like boxes. Twist is known to activate FGFR and
NK-homeodomain target genes during mesodermal patterning of Drosophila; similar target interactions
have been proposed to modulate mesenchymal growth during closure of the vertebrate skull. These results
suggest the possibility that a conserved pathway may be used for diverse functions in mesodermal
specification (Harfe, 1998).
Saethre-Chotzen syndrome is one of the most common autosomal dominant disorders of
craniosynostosis in humans and is characterized by craniofacial and limb anomalies. The locus for
Saethre-Chotzen syndrome maps to chromosome 7p21-p22. TWIST (The human homolog of Drosophila Twist) has been evaluated as a candidate gene for this condition because its expression
pattern and mutant phenotypes in Drosophila and mouse are consistent with the Saethre-Chotzen
phenotype. TWIST maps to human chromosome 7p21-p22 and mutational analysis reveals
nonsense, missense, insertion and deletion mutations in patients. These mutations occur within the basic
DNA binding, helix I and loop domains, or result in premature termination of the protein. Studies in
Drosophila indicate that twist may affect the transcription of fibroblast growth factor receptors
(FGFRs), another gene family implicated in human craniosynostosis. The emerging cascade of
molecular components involved in craniofacial and limb development now includes TWIST, which may
function as an upstream regulator of FGFRs (Howard, 1997).
Mutually exclusive use of exons IIIb or IIIc in FGF-R2 transcripts requires the silencing of exon IIIb. This repression is mediated by silencer elements upstream and downstream of the exon. Both silencers bind the polypyrimidine tract binding protein (PTB: see Drosophila Hephaestus) and PTB binding sites within these elements are required for efficient silencing of exon IIIb. Recruitment of MS2-PTB fusion proteins upstream or downstream of exon IIIb causes repression of this exon. Depletion of endogenous PTB using RNAi increases exon IIIb inclusion in transcripts derived from minigenes and from the endogenous FGF-R2 gene. These data demonstrate that PTB is a negative regulator of exon definition in vivo (Wagner, 2002).
Deletion of PTB binding sites and knockdown of PTB both leads to an approximately 3-fold increase in exon IIIb inclusion, whereas deletion of a complete intronic control element leads to a 10-fold increase in exon inclusion. The most likely explanation for these results is that PTB collaborates with other unidentified factors, which bind to the 5' control element. The need for multiple factors to bind adjacent elements to integrate an alternative splicing outcome has been noted in several cases. HnRNP H, hnRNP F, KSRP, and nPTB have been found to bind the downstream splicing enhancer, which is required for inclusion of the N1 in c-src mRNAs in neural tissues. The tissue-specific inclusion of the alternative exon 5 of cardiac troponin-T appears to require the integrated activity of PTB and members of the msl family of factors. PTB associates with FBP and Sam68 on the intron upstream of the regulated exon 7 in rat ß-tropomyosin transcripts. The need to regulate a vast number of alternative splicing events has been solved by the integration of the activity of a limited number of factors that by combinatorial assortment can lead to very large number of functional states. An elegant binary switch provided by a single alternative splicing factor, as is the case for Sxl protein in D. melanogaster, may be reserved for crucial decisions, such as sex determination, which are made very early in development (Wagner, 2002).
It is clear that the factors that mediate silencing of exon IIIb via the upstream intronic splicing silencer and the intronic control element are present and active in both fibroblasts and epithelial cells. How then is exon IIIb included in FGF-R2 mRNAs in epithelial cells? It is likely that a cell type-specific factor or perhaps a combination of factors results in the specific derepression of exon IIIb. Exon IIIb activating factors are recruited via the intronic activating sequence 2 and the upstream activating element ISAR; these sites work in concert and form a secondary structure that is required for their function. Given that intronic activating sequence 2 is embedded within intronic element, it is reasonable to predict that the IAS2-ISAR structure will disrupt the silencing topology. Thus exon IIIb inclusion in epithelial cells is likely achieved by countering the repression mechanism instituted by PTB and other yet unidentified splicing repressors (Wagner, 2002).
Fibroblast growth factor (FGF) receptors trigger a wide variety of cellular responses as diverse as cell migration, cell proliferation and cell differentiation. However, the molecular basis of the specificity of these responses is not well understood. The C. elegans FGF receptor EGL-15 similarly mediates a number of different responses, including transducing a chemoattractive signal and mediating an essential function. Analysis of the migration-specific alleles of egl-15 has identified a novel EGL-15 isoform that provides a molecular explanation for the different phenotypic effects of lesions at this locus. Alternative splicing yields two EGL-15 proteins containing different forms of a domain located within the extracellular region of the receptors immediately after the first IG domain. Neither of these two domain forms is found in any other FGF receptor. The roles of these EGL-15 receptor isoforms and their two FGF ligands were examined for their signaling specificity. These analyses demonstrate different physiological functions for the two receptor variants. EGL-15(5A) is required for the response to the FGF chemoattractant that guides the migrating sex myoblasts to their final positions. By contrast, EGL-15(5B) is both necessary and sufficient to elicit the essential function mediated by this receptor (Goodman, 2003).
FGFR1 is an important signalling molecule during embryogenesis and in adulthood. FGFR1 mutations in human may lead to developmental defects and pathological conditions, including cancer and Alzheimer's disease. Cloning and expression analysis of the zebrafish fibroblast growth factor receptor 1 (fgfr1) is described. Initially, fgfr1 is expressed in the adaxial mesoderm with transcripts distinctly localised to the anterior portion of each half-somite. Hereupon, fgfr1 is also strongly expressed in the otic vesicles, branchial arches and the brain, especially at the midbrain-hindbrain boundary (MHB). The expression patterns of fgfr1 and fgf8 are strikingly similar and knock-down of fgfr1 phenocopies many aspects observed in the fgf8 mutant acerebellar, suggesting that Fgf8 exerts its function mainly by binding to FgfR1 (Scholpp, 2004).
Mice with the K644E kinase domain mutation in fibroblast growth factor receptor 3 (Fgfr3) (EIIa;Fgfr3+/K644E) exhibit a marked enlargement of the brain. The brain size is increased as early as E11.5, not secondary to the possible effect of Fgfr3 activity in the skeleton. Furthermore, the mutant brains show a dramatic increase in cortical thickness, a phenotype opposite that in FGF2 knockout mice. Despite this increased thickness, cortical layer formation is largely unaffected and no cortical folding is observed during embryonic days 11.5-18.5 (E11.5-E18.5). Measurement of cortical thickness revealed an increase of 38.1% in the EIIa;Fgfr3+/K644E mice at E14.5 and an advanced appearance of the cortical plate was frequently observed at this stage. Unbiased stereological analysis revealed that the volume of the ventricular zone (VZ) is increased by more than two fold in the EIIa;Fgfr3+/K644E mutants at E14.5. A relatively mild increase in progenitor cell proliferation and a profound decrease in developmental apoptosis during E11.5-E14.5 most likely accounts for the dramatic increase in total telecephalic cell number. Taken together, these data suggest a novel function of Fgfr3 in controlling the development of the cortex, by regulating proliferation and apoptosis of cortical progenitors (Inglis-Broadgate, 2005).
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