FGF receptor 1
FGF receptor, somitogenesis, myogenesis, cardiogenesis, chondrogenesis and bone development The sex myoblasts (SMs) in C. elegans hermaphrodites undergo anteriorly directed cell migrations
that allow for the proper localization of the muscles involved in egg-laying. These migrations are controlled in
part by a signal emanating from gonadal cells that allows the SMs to be attracted to their precise
final positions flanking the center of the gonad. Mutations in egl-15 alter the nature of the interaction
between the gonad and the SMs, resulting in the posterior displacement of the SMs. egl-15 encodes a receptor tyrosine kinase of the fibroblast growth factor receptor (FGFR)
subfamily, capable of playing multiple roles in development. Three genes have been identified that behave genetically as
activators or mediators of egl-15 activity. One of these,sem-5, encodes an adaptor molecule
that transduces signals from a variety of receptor tyrosine kinases (DeVore, 1995).
A gene encoding a fibroblast growth factor
receptor (SpFGFR) is transcribed in many cell types during the initial phases of sea urchin embryogenesis
(Strongylocentrotus purpuratus). SpFGFR protein is detectable only in
muscle cells of the embryo and appears at a time suggesting that its function is not in commitment to a
muscle fate, but instead may be required to support the proliferation, migration, and/or differentiation of
myoblasts. SpFGFR transcripts are enriched in embryo nuclei, suggesting that
lack of processing and/or cytoplasmic transport in nonmuscle cells is at least part of the posttranscriptional
regulatory mechanism. SpFGFR is also specifically expressed in adult lantern
muscle, but is not detectable in other smooth muscle-containing tissues, including tube foot and intestine,
or in coelomocytes, despite the presence of SpFGFR transcripts at similar concentrations in all these
tissues. It is concluded that in both embryos and adults, muscle-specific SpFGF receptor synthesis is
controlled primarily at a posttranscriptional level. Transcripts
encoding the IgS variant of the ligand binding domain of the receptor, previously shown to be enriched in
embryo endomesoderm fractions, are the predominant, if not exclusive, form of SpFGFR transcripts in lantern
muscle. Together, these results suggest that only a minority of SpFGFR transcripts are processed,
exported, and translated in both adult and embryonic muscle cells and these contain predominantly, if not
exclusively, IgS ligand binding domain sequences (McCoon, 1998).
An avian FGF receptor, FREK, is expressed by replicating skeletal muscle myoblasts,
while differentiated muscle cells no longer express this receptor. In the limb, muscle progenitors
originating from the somite start expressing FREK at 3 days of development (E3). FREK
expression in the limb myoblasts follows that of Pax-3 (Drosophila homolog: Paired) and Pax-7, but precedes that of MyoD.
Since MyoD expression signals the onset of terminal differentiation, this demonstrates that FREK is
expressed in muscle progenitors prior to overt muscle differentiation. A more complex situation is
observed in the trunk region, where a first wave of MyoD-positive myocytes, which are postmitotic
and never express FREK, appear in the early myotomal compartment of the somite. Slightly later,
at E2.5, FREK-positive myoblasts migrate into the myotome, constituting a second wave of muscle
progenitors, 15 hr after the first MyoD-positive cells. FREK's expression by myoblasts arising at all
stages of myogenesis indicates that this growth factor receptor represents one of the earliest
molecular markers for this cell population. FREK's prominent expression during muscle
differentiation sets it apart from other FGF receptors and suggests that this molecule plays an
important role during muscle growth and differentiation (Marcelle, 1995).
Epithelial-mesenchymal interactions are of major importance during development to direct
correct differentiation and morphogenesis of embryonic tissues. One subset of lateral
mesoderm-derived mesenchymal cells will form the smooth muscle (SM) layer of the primary
epithelial lining of hollow internal organs. The differentiation
of SM cells in Xenopus can be followed by the expression of alpha-SM actin. Basic fibroblast growth factor (bFGF) has the ability to induce this actin isoform in
isolated blastula animal pole cells. Messenger RNA encoding
a truncated form of the FGF receptor can act as a dominant negative inhibitor and results in significant delay in the differentiation of the SM cells
compared to control embryos, demonstrating the importance of this signaling pathway for the
formation of the lateral mesoderm-derived SM cells. Moreover, a correlation exists between this delay and the dramatic defects observed in the morphogenesis of the
intestine with which mesenchyme-derived SM cells are normally associated. This phenotype is
efficiently rescued by coinjection of the wild-type FGF receptor. The timely
differentiation of SM cells is an essential event for the proper
morphogenesis of the endoderm-derived digestive tract (Saint-Jeannet, 1994).
To investigate the roles of FGF signaling in the regulation of myogenesis both in the somite
and the limb bud, mosaic chicken embryos were generated that consist of somitic cells carrying
transgenes expressing one of the following: FGF1, FGF4, the FGF receptor type-1 (FGFR1) or its
dominant negative mutant (delta FGFR1). Cells producing FGF ligand migrate
into the somatopleure without differentiating into myotomal muscle, but differentiate into muscle
fibers when they arrive in the limb bud. In contrast, cells overexpressing FGFR1 migrate into the
limb muscle mass but remain as undifferentiated myoblasts. Cells expressing the delta
FGFR1 fail to migrate to the somatopleure but are capable of differentiating
into myotomal muscle within the somites. These results suggest that the FGFR-mediated FGF
signaling (1) blocks terminal differentiation of myogenic cells within the somite and (2) sustains
myoblast migration to limb buds from the somite, and that (3) down-regulation of FGFRs or FGFR
signaling is involved in mechanisms triggering terminal differentiation of the limb muscle mass during
avian embryogenesis (Itoh, 1996).
In birds and mammals, cardiac myocytes terminate mitotic activity in the neonatal period;
regeneration of cardiac muscle does not occur after myocardial injury in adult hearts. Even
embryonic myocytes, which actively proliferate in vivo, quickly lose mitotic activity when placed
in cell culture. Several growth factors, including fibroblast growth factor (FGF), have been
documented in embryonic hearts and some have been shown to influence myocyte terminal
differentiation in culture. However, none of these growth factors have been shown to reactivate
cell division in postmitotic myocytes nor have their in vivo functions been defined satisfactorily.
To clarify the role of FGF signaling in heart growth, a dominant-negative mutant of
receptor type 1 (FGFR1) and FGFR1
antisense RNA inhibit myocyte proliferation and/or survival during the first week
of chicken embryonic development but had much less effect after the second week. No
apparent alteration of myocyte growth is observed after overexpression of full-length FGFR1.
These results suggest that receptor-coupled FGF signaling regulates cardiac myocyte growth
during tubular stages of cardiogenesis but that myocyte growth becomes FGF-independent after
the second week of embryogenesis (Mima, 1995).
Fibroblast growth factor (FGF) stimulates proliferation
and represses differentiation of a myoblast cell line. The predominant FGF receptor present on MM14
cells, FGFR1 (Drosophila homolog: FGFR1), is a receptor tyrosine kinase capable of activating the mitogen-activated protein
kinase (MAPK) cascade in fibroblast and neuronal cell lines. To determine whether the FGF
signal is mediated via the MAPK cascade in myoblasts, MM14 cells were stimulated with basic
FGF (bFGF) and activities of the various kinases were measured. After withdrawal from serum
and bFGF for 3 hr, bFGF stimulates MAPK kinase (MAPKK) activity, but MAPK (Drosophila homolog: Rolled/MAPK) and S6
peptide kinase activities were not detected. In contrast, when serum and bFGF were withdrawn
for 10 hr, the activities of MAPKK, MAPK, and S6 peptide kinase are all stimulated by
bFGF treatment. The inability of bFGF to stimulate MAPK after 3 hr of withdrawal may be due,
in part, to the presence of a MAPK phosphatase activity that was detected in MM14 cell
extracts. This dephosphorylating activity diminishes during commitment to terminal differentiation
and is inhibited by sodium orthovanadate. Thus, the ability of bFGF to stimulate MAPK in
MM14 cells is correlated with the loss of a MAPK phosphatase activity. These results show
that although bFGF activates MAPKK in proliferating myoblasts, the mitogenic signal does not
progress to the downstream kinases, providing a physiological example of an uncoupling of the
MAPK cascade (Campbell, 1995).
Ligand-stimulated activation of FGF receptors (FGFRs) in skeletal muscle cells represses terminal myogenic differentiation.
Skeletal muscle cell lines and subsets of primary cells are dependent on FGFs to repress myogenesis and maintain growth. To
understand the intracellular events that transduce these signals, MM14 skeletal muscle cells were transfected with expression
vectors encoding chimeric receptors. The chimeras are comprised of the PDGF beta receptor (PDGFbetaR) extracellular domain,
the FGFR-1 intracellular domain, and either the PDGFbetaR or FGFR-1 transmembrane domain. The chimeric receptors are
autophosphorylated upon PDGF-BB stimulation and are capable of stimulating mitogen-activated protein kinase activity.
Activation of the tyrosine kinase domain of either chimera represses myogenesis, suggesting that intracellular responses regulating
skeletal muscle differentiation are transduced by activation of the FGFR-1 tyrosine kinase. Activation
of either chimeric receptor fails to stimulate cellular proliferation. Thus, it appears that regulation of skeletal muscle differentiation
by FGFs requires only activation of the FGFR tyrosine kinase. In contrast, stimulation of proliferation may require additional, as
yet unidentified, signals involving the receptor ectodomain, the FGF ligand, and heparan sulfate either alone, or in combination (Kudla, 1998).
The identities of extracellular growth factors that regulate skeletal muscle development in vivo are largely unknown. It was
asked if FGFs, which act as repressors of myogenesis in culture, play a similar role in vivo by ectopically expressing in the
developing limb a truncated FGF receptor 1 (dnFGFR1) that acts as a dominant negative mutant. Hind limbs and the
adjacent somites of stage 17 chickens were infected with a replication-competent RCAS virus encoding dnFGFR1. By ED5, the virus had spread extensively within the limb and the adjacent somites with little
rostral or caudal expansion of the infection along the axial midline. Viral infection and mutant receptor expression were
coincident as revealed by the distribution of a viral coat protein and an HA epitope tag present on the carboxy terminus of
dnFGFR1. Within 48 h following injection of dnFGFR1, no obvious changes in skeletal muscle precursor
cell migration into the hind limb could be detected as compared to control limbs infected with an empty RCAN virus. However, by 3 days
following infection of RCAS-dnFGFR1 virus, the level of skeletal muscle-specific myosin heavy chain was decreased and the
expression pattern altered, suggesting disruption of skeletal muscle development. Two striking muscular phenotypes were
observed in dnFGFR1-expressing limbs, including an average loss of 30% in skeletal muscle wet weight and a 50% decrease
in myofiber density. At all ages examined the loss of skeletal muscle mass was accompanied by a loss of myoblasts and an
unexpected concomitant loss of fibroblasts. Consistent with these observations, explants of infected cells revealed a
reduction in the number of myonuclei in myotubes. Although the myofiber density per unit area was decreased over 50%
compared to controls, there were no detectable effects on myofiber diameter. The loss in myofiber density was, however,
accompanied by an increase in the space surrounding individual myofibers and a generalized loss of myofiber integrity. It
is noteworthy that long-bone development is unaffected by RCAS-dnFGFR1 infection, suggesting that FGFR2 and FGFR3
signaling is not disrupted. These data provide conclusive evidence that FGFR1 signaling is necessary to maintain myoblast
number and this signaling plays a role in myofiber organization (Flanagan-Steet, 2000).
Fibroblast growth factor receptors 1 and 3 have distinct mitogenic activities in vitro. In several cultured cell lines, FGFR1 transmits a potent mitogenic signal, whereas FGFR3 has little or no mitogenic activity. However, in other in vitro assays the FGFR3 intracellular domain is comparable with that of FGFR1. In vivo, FGFR3 negatively regulates chondrocyte proliferation and differentiation, and activating mutations are the molecular etiology of achondroplasia. By contrast, FGFR1 transmits a proliferative signal in various cell types in vivo. These observations suggest that inhibition of the proliferating chondrocyte could be a unique property of FGFR3 or, alternatively, a unique property of the proliferating chondrocyte. To test this hypothesis, FGFR1 signaling was activated in the growth plate in cells that normally express FGFR3. Comparison of transgenic mice with an activated FGFR1 signaling pathway with an achondroplasia-like mouse that expresses a similarly activated FGFR3 signaling pathway has demonstrated that both transgenes result in a similar achondroplasia-like dwarfism. These data demonstrate that suppression of mitogenic activity by FGFR signaling is a property that is unique to growth plate chondrocytes. Surprisingly, it was observed that in transgenic mice expressing an activated FGFR, some synovial joints fail to develop and are replaced by cartilage. The defects in the digit joints phenocopy the symphalangism that occurs in Apert syndrome and the number of affected joints is dependent on transgene dose. In contrast to the phenotype in the growth plate, the joint phenotype is more severe in transgenic mice with an activated FGFR1 signaling pathway. The failure of joint development results from expanded chondrification in the presumptive joint space, suggesting a crucial role for FGF signaling in regulating the transition of condensed mesenchyme to cartilage and in defining the boundary of skeletal elements (Wang, 2001).
Fibroblast growth factor receptor type 2 (FGFR2) plays major roles in development. Like FGFR1 and FGFR3, it exists as two splice variants, IIIb and IIIc. The function of FGFR2IIIc, the mesenchymal splice variant of FGFR2, has been investigated in the mouse. Fgfr2IIIc is expressed in early mesenchymal condensates and in the periosteal collar around the cartilage models; later it is expressed in sites of both endochondral and intramembranous ossification. A translational stop codon inserted into exon 9 disruptes the synthesis of Fgfr2IIIc without influencing the localized transcription of Fgfr2IIIb, the epithelial Fgfr2 variant. The recessive phenotype of Fgfr2IIIc–/– mice is characterized initially by delayed onset of ossification, with continuing deficiency of ossification in the sphenoid region of the skull base. During subsequent stages of skeletogenesis, the balance between proliferation and differentiation is shifted towards differentiation, leading to premature loss of growth, synostosis in certain sutures of the skull base and in the coronal suture of the skull vault, with dwarfism in the long bones and axial skeleton. The retarded ossification is correlated with decrease in the localized transcription of the osteoblast markers secreted phosphoprotein 1 (Spp1) and Runx2/Cbfa1. A decrease in the domain of transcription of the chondrocyte markers Ihh and PTHrP (Pthlh) corresponds with a decrease in their transcripts in the proliferative and hypertrophic chondrocyte zones. These results suggest that Fgfr2IIIc is a positive regulator of ossification affecting mainly the osteoblast, but also the chondrocyte, lineages. This role contrasts with the negative role of Fgfr3, although recent reports implicate FGF18, a ligand for FGFR3IIIc and FGFR2IIIc, as a co-ordinator of osteogenesis via these two receptors (Eswarakumar, 2002).
The formation of cartilage elements in the developing vertebrate limb, where
they serve as primordia for the appendicular skeleton, is preceded by the appearance of discrete cellular condensations. Control of the size and spacing of these condensations is a key aspect of skeletal pattern formation. Limb bud cell cultures grown in the absence of ectoderm form continuous sheet-like masses of cartilage. With the inclusion of ectoderm, these cultures produce one or more cartilage nodules surrounded by zones of noncartilaginous mesenchyme. Ectodermal fibroblast growth factors (FGF2 and FGF8), but
not a mesodermal FGF (FGF7), substitute for ectoderm in inhibiting chondrogenic gene expression, with some combinations of the two ectodermal factors leading to well-spaced cartilage nodules of relatively uniform size. Treatment
of cultures with SU5402, an inhibitor FGF receptor tyrosine kinase activity, renders FGFs ineffective in inducing perinodular inhibition. Inhibition of production of FGF receptor 2 (FGFR2) by transfection of wing and leg cell cultures with antisense oligodeoxynucleotides, blocks appearance of ectoderm- or FGF-induced zones of perinodular inhibition of chondrogenesis, and, when introduced into the limb buds of developing embryos, this leads to shorter, thicker, and fused cartilage elements. Because FGFR2 is expressed mainly at sites of precartilage condensation during limb development in vivo and in vitro, these results suggest that activation of FGFR2 by FGFs during development elicits a lateral inhibitor of chondrogenesis that limits the expansion of developing skeletal elements (Moftah, 2002).
Human craniosynostosis syndromes, resulting from activating or neomorphic mutations in fibroblast growth factor receptor 2 (FGFR2), underscore an essential role for FGFR2 signaling in skeletal development. Embryos harboring homozygous null mutations in FGFR2 die prior to skeletogenesis. To address the role of FGFR2 in normal bone development, a conditional gene deletion approach was adopted. Homologous introduction of cre recombinase into the Dermo1 (Twist2) gene locus results in robust expression of CRE in mesenchymal condensations giving rise to both osteoblast and chondrocyte lineages. Inactivation of a floxed Fgfr2 allele with Dermo1-cre results in mice with skeletal dwarfism and decreased bone density. Although differentiation of the osteoblast lineage is not disturbed, the proliferation of osteoprogenitors and the anabolic function of mature osteoblasts are severely affected (Yu, 2003).
Fibroblast growth factor receptor signaling is an important mechanism regulating osteoblast function. To gain an insight into the regulatory role of FGF receptor-2 (FGFR2) signaling in osteoblasts, integrin-mediated attachment and cell survival were investigated in human calvarial osteoblasts expressing activated FGFR2. FGFR2 activation reduces osteoblast attachment on fibronectin. This is associated with reduced expression of the alpha5 integrin subunit normally expressed in human calvarial osteoblasts in vivo. Treatment with lactacystin, a potent inhibitor of proteasome, restores alpha5 integrin levels in FGFR2 mutant osteoblasts. Immunoprecipitation analysis shows that alpha5 integrin interacts with both the E3 ubiquitin ligase Cbl and ubiquitin. Immunocytochemistry revealed that alpha5 integrin colocalizes with FGFR2 and Cbl at the leading edge in membrane ruffle regions. Transfection with the 70Z-Cbl mutant lacking the RING domain required for Cbl-ubiquitin interaction, or with the G306E Cbl mutant that abolishes the binding ability of Cbl phosphotyrosine-binding domain restores alpha5 integrin levels. This suggests that Cbl-mediated ubiquitination plays an essential role in alpha5 integrin proteasome degradation induced by FGFR2 activation. Reduced alpha5 integrin expression is associated with an increased Bax/Bcl-2 ratio and increased caspase-9 and -3 activities in FGFR2 mutant osteoblasts. Forced expression of alpha5 integrin rescues cell attachment and corrects both the Bax/Bcl-2 ratio and caspase-3 and caspase-9 activities in FGFR2 mutant osteoblasts. Cbl recruitment induced by FGFR2 activation triggers alpha5 integrin degradation by the proteasome, which results in reduced osteoblast attachment on fibronectin and caspase-dependent apoptosis. This identifies a functional role of the alpha5 integrin subunit in the induction of apoptosis triggered by FGFR2 activation in osteoblasts, and reveals that a Cbl-dependent mechanism is involved in the coordinated regulation of cell apoptosis induced by alpha5 integrin degradation (Kaabeche, 2005).
Bone morphogenetic protein (BMP) signaling pathways are essential
regulators of chondrogenesis. However, the roles of these pathways in vivo are
not well understood. Limb-culture studies have provided a number of essential
insights, including the demonstration that BMP pathways are required for
chondrocyte proliferation and differentiation. However, limb-culture studies
have yielded contradictory results; some studies indicate that BMPs exert
stimulatory effects on differentiation, whereas others support inhibitory
effects. Therefore, this study characterized the skeletal phenotypes of mice lacking Bmpr1a in chondrocytes (Bmpr1aCKO) and
Bmpr1aCKO;Bmpr1b+/-
(Bmpr1aCKO;1b+/-) in order to test the roles of
BMP pathways in the growth plate in vivo. These mice reveal requirements for
BMP signaling in multiple aspects of chondrogenesis. They also demonstrate
that the balance between signaling outputs from BMP and fibroblast growth
factor (FGF) pathways plays a crucial role in the growth plate. These studies
indicate that BMP signaling is required to promote Ihh expression,
and to inhibit activation of STAT and ERK1/2 MAPK, key effectors of FGF
signaling. BMP pathways inhibit FGF signaling, at least in part, by inhibiting
the expression of FGFR1. These results provide a genetic in vivo demonstration
that the progression of chondrocytes through the growth plate is controlled by
antagonistic BMP and FGF signaling pathways (Yoon, 2006).
Fibroblast growth factor (FGF) signaling plays a crucial role in vertebrate segmentation. The FGF pathway establishes a posterior-to-anterior signaling gradient in the presomitic mesoderm (PSM), which controls cell maturation and is involved in the positioning of segmental boundaries. In addition, FGF signaling was shown to be rhythmically activated in the PSM in response to the segmentation clock. This study shows that conditional deletion of the FGF receptor gene Fgfr1 abolishes FGF signaling in the mouse PSM, resulting in an arrest of the dynamic cyclic gene expression and ultimately leading to an arrest of segmentation. Pharmacological treatments disrupting FGF signaling in the PSM result in an immediate arrest of periodic WNT activation, whereas Notch-dependent oscillations stop only during the next oscillatory cycle. Together, these experiments provide genetic evidence for the role of FGF signaling in segmentation, and identify a signaling hierarchy controlling clock oscillations downstream of FGF signaling in the mouse (Wahl, 2007).
Achondroplasias are the most common genetic forms of dwarfism in humans. They are associated with activating mutations in FGFR3, which signal through the Stat and MAPK pathways in a ligand-independent manner to impair chondrocyte proliferation and differentiation. Snail1 has been implicated in chondrocyte differentiation as it represses Collagen II and aggrecan transcription in vitro. Snail1 overexpression in the developing bone leads to achondroplasia in mice. Snail1 acts downstream of FGFR3 signaling in chondrocytes, regulating both Stat and MAPK pathways. Moreover, FGFR3 requires Snail1 during bone development and disease as the inhibition of Snail1 abolishes its signaling even through achondroplastic- and thanatophoric-activating FGFR3 forms. Significantly, Snail1 is aberrantly upregulated in thanatophoric versus normal cartilages from stillborns. Thus, Snail activity may likely be considered a target for achondroplasia therapies (de Frutos, 2007).
In order to understand how secreted signals regulate complex morphogenetic events, it is crucial to identify their cellular targets. By conditional inactivation of Fgfr1 and Fgfr2 and overexpression of the FGF antagonist sprouty 2 in different cell types, the role of FGF signaling was dissected during heart outflow tract development in mouse. Contrary to expectation, cardiac neural crest and endothelial cells are not primary paracrine targets. FGF signaling within second heart field mesoderm is required for remodeling of the outflow tract: when disrupted, outflow myocardium fails to produce extracellular matrix and TGFbeta and BMP signals essential for endothelial cell transformation and invasion of cardiac neural crest. It is concluded that an autocrine regulatory loop, initiated by the reception of FGF signals by the mesoderm, regulates correct morphogenesis at the arterial pole of the heart. These findings provide new insight into how FGF signaling regulates context-dependent cellular responses during development (Park, 2008).
In contrast to the paradigm of paracrine signaling established in other tissues, these data show that in pharyngeal and splanchnic mesoderm dorsal to the heart tube, called the second heart field, the cellular source of the ligand (signal) is also the target. Such an autocrine pathway can be easily understood in terms of a feedback loop that maintains FGF production within a tight range, which is crucial for FGF8 function. Secondary effects on other signaling pathways that were observed may also be integrated into this regulatory loop. Fgf8 and FGFR mutant analyses establish that the autocrine pathway not only regulates survival and proliferation of second heart field cells (a common response to FGFs), but also the secretory and signaling capacities of their derivatives in the arterial pole of the heart, called the outflow tract. The few transcriptional targets of the PEA3 family of FGF8 effector proteins thus far identified are ECM components, ECM-modifying enzymes and cell adhesion molecules, suggesting that an autocrine pathway might provide a means of regulating the ECM and microenvironment to ensure uniform signal reception and response within a specialized cell population. These findings are of biomedical importance, not only in the context of understanding the causes of congenital malformations of the outflow tract, but also because the crucial role demonstrated for an autocrine FGF signaling pathway has broad implications for understanding fundamental properties of FGF signaling in different developmental and pathological contexts (Park, 2008).
The cardiac outflow tract (OFT) is a developmentally complex structure derived from multiple lineages and is often defective in human congenital anomalies. Although emerging evidence shows that fibroblast growth factor (FGF) is essential for OFT development, the downstream pathways mediating FGF signaling in cardiac progenitors remain poorly understood. This study reports that FRS2alpha (FRS2), an adaptor protein that links FGF receptor kinases to multiple signaling pathways, mediates crucial aspects of FGF-dependent OFT development in mouse. Ablation of Frs2alpha in mesodermal OFT progenitor cells that originate in the second heart field (SHF) affects their expansion into the OFT myocardium, resulting in OFT misalignment and hypoplasia. Moreover, Frs2alpha mutants have defective endothelial-to-mesenchymal transition and neural crest cell recruitment into the OFT cushions, resulting in OFT septation defects. These results provide new insight into the signaling molecules downstream of FGF receptor tyrosine kinases in cardiac progenitors (Zhang, 2008).
FGF receptors, hematopoiesis, and angiogenesis BLast Colony Forming Cells (BL-CFCs)
from in vitro differentiated embryonic stem (ES)
cells represent the common progenitor of hematopoietic
and endothelial cells, the hemangioblast. Access to this
initial cell population committed to the hematopoietic
lineage provides a unique opportunity to characterize
hematopoietic commitment events. Here, BL-CFC
expresses the receptor tyrosine kinase, Flk1 (the VEGF receptor), and thus
advantage was taken of the BL-CFC assay, as well as
fluorescent activated cell sorter (FACS) analysis for Flk1 +
cells to determine quantitatively if mesoderm-inducing
factors promote hematopoietic lineage development.
Moreover, ES lines carrying targeted
mutations for fibroblast growth factor receptor-1 (fgfr1), a
receptor for basic fibroblast growth factor (bFGF), as well
as scl, a transcription factor, were analyzed for their potential to generate
BL-CFCs and Flk1 + cells, to further define events leading
to hemangioblast development. bFGF
together with activin A appear to have an additive or
synergistic effect on BL-CFC generation; activin A-mediated
BL-CFC generation requires a Fgfr1 signal since the
administration of activin A does not rescue the BL-CFC
development in fgfr1-/- embryoid bodies [in vitro
differentiated progeny (embryoid bodies, EBs) of embryonic
stem (ES) cells]. These results are
consistent with the findings in Xenopus that activin A and
bFGF synergize in mesoderm induction and that activin A
requires a bFGF-mediated signal. The data suggest that
bFGF-mediated signaling is critical for the proliferation of
the hemangioblast and that cells expressing both Flk1 and
SCL may represent the hemangioblast (Faloon, 2000).
Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is an essential physiological process in
development, yet also plays a major role in the progression of human diseases such as diabetic retinopathy, atherosclerosis and
cancer. The effects of the most potent angiogenic factors -- vascular endothelial growth factor (VEGF), angiopoietin and
fibroblast growth factor (FGF) -- are mediated through cell surface receptors that possess intrinsic protein tyrosine kinase
activity. This report describes a synthetic compound of the pyrido[2,3-d]pyrimidine class, designated PD 173074 that
selectively inhibits the tyrosine kinase activities of the FGF and VEGF receptors. Systemic administration of PD
173074 in mice can effectively block angiogenesis induced by either FGF or VEGF with no apparent toxicity. To elucidate the
determinants of selectivity, the crystal structure of PD 173074 was determined in complex with the tyrosine kinase domain
of FGF receptor 1 at 2.5 A resolution. A high degree of surface complementarity between PD 173074 and the hydrophobic,
ATP-binding pocket of FGF receptor 1 underlies the potency and selectivity of this inhibitor. PD 173074 is thus a promising
candidate for a therapeutic angiogenesis inhibitor to be used in the treatment of cancer and other diseases whose progression is
dependent on new blood vessel formation (Mohammadi, 1998).
Ligand-dependent signalling pathways have been characterized as having
morphogen properties where there is a quantitative relationship between
receptor activation and response, or threshold characteristics in which there
is a binary switch in response at a fixed level of receptor activation. In this study a bacterial artificial chromosome (BAC)-based transgenic
system was used in which a hypermorphic mutation was introduced into the murine
Fgfr1 gene. These mice exhibit cranial suture and sternal fusions
that are exacerbated when the BAC copy number is increased. Surprisingly,
increasing mutant BAC copy number also leads to the de novo appearance of
digit I polydactyly in the hind limb and transformations of the vertebrae.
Polydactyly is accompanied by a reduction of programmed cell death in the
developing hind limb. Candidate gene analysis reveals downregulation of
Dkk1 in the digit I field and upregulation of Wnt5a and
Hoxd13. These findings show that Fgfr1-mediated developmental
pathways exhibit differing signalling dynamics, whereby development of the
cranial sutures and sternum follows a morphogen mode, whereas development of
the vertebral column and the hind limbs has threshold signalling
properties (Hajihosseini, 2004).
Blood and lymphatic vasculatures are intimately involved in tissue oxygenation and fluid homeostasis maintenance. Assembly of these vascular networks involves sprouting, migration and proliferation of endothelial cells. Recent studies have suggested that changes in cellular metabolism are important to these processes. Although much is known about vascular endothelial growth factor (VEGF)-dependent regulation of vascular development and metabolism, little is understood about the role of fibroblast growth factors (FGFs) in this context. This study identified FGF receptor (FGFR; see Drosophila Breathless) signalling as a critical regulator of vascular development. This is achieved by FGF-dependent control of c-MYC (MYC; see Drosophila Myc) expression that, in turn, regulates expression of the glycolytic enzyme hexokinase 2 (HK2; see Drosophila Hexokinase A). A decrease in HK2 levels in the absence of FGF signalling inputs results in decreased glycolysis, leading to impaired endothelial cell proliferation and migration. Pan-endothelial- and lymphatic-specific Hk2 knockouts phenocopy blood and/or lymphatic vascular defects seen in Fgfr1/Fgfr3 double mutant mice, while HK2 overexpression partly rescues the defects caused by suppression of FGF signalling. Thus, FGF-dependent regulation of endothelial glycolysis is a pivotal process in developmental and adult vascular growth and development (Yu, 2017).
Based on data from in vitro tissue explant and ex vivo cell/bead
implantation experiments, Bmp and Fgf signaling have been proposed to regulate
hepatic specification. However, genetic evidence for this hypothesis has been
lacking. This study provides in vivo genetic evidence that Bmp and Fgf signaling
are essential for hepatic specification. Transgenic zebrafish were used that
overexpress dominant-negative forms of Bmp or Fgf receptors following
heat-shock induction. These transgenes allow one to bypass the early embryonic
requirements for Bmp and Fgf signaling, and also to completely block Bmp or
Fgf signaling. It was found that the expression of hhex and
prox1, the earliest liver markers in zebrafish, was severely reduced
in the liver region when Bmp or Fgf signaling was blocked just before hepatic
specification. However, hhex and prox1 expression in
adjacent endodermal and mesodermal tissues appeared unaffected by these
manipulations. Additional genetic studies indicate that the endoderm maintains
competence for Bmp-mediated hepatogenesis over an extended window of embryonic
development. Altogether, these data provide the first genetic evidence that
Bmp and Fgf signaling are essential for hepatic specification, and suggest
that endodermal cells remain competent to differentiate into hepatocytes for
longer than anticipated (Shin, 2007).
FGF receptors and skull development The FGFR2 gene is of particular interest in the context of craniofacial development. Dominantly acting missense mutations located mainly in the IgIIIa/IIIc domain or in the IgII/IgIII linker region are associated with a variety of craniosynostosis syndromes. Common to all phenotypes is the consistency with which the coronal suture, as compared to other sutures, shows premature fusion. Some of these syndromes, as well as non-syndromic coronal craniosynostosis, have also been found to be associated with equivalent mutations of FGFR1 or FGFR3. Abnormal FGFR signaling has also been implicated in the coronal craniosynostosis seen in Saethre-Chotzen syndrome, which is due to a mutation of twist (Drosophila homolog: twist): the twist gene product is a transcription factor that appears to be a prerequisite for FGFR signaling during mesoderm formation, required for the expression of FGFRs. Many of these FGFR-related craniosynostosis syndromes additionally show abnormalities of the digits of the hands and feet, but achondroplasia and related syndromes affecting the growth plates of long-bones have to date only been associated with mutation of FGFR3 (Iseki, 1997 and references).
In the fetal mouse skull vault, Fgfr2 transcripts are most abundant at the periphery of the
membrane bones; they are mutually exclusive with those of osteopontin (an early marker of osteogenic differentiation) but coincide
with sites of rapid cell proliferation. Fibroblast growth factor type 2 (FGF2) protein, which has a high affinity for the FGFR2 splice
variant associated with craniosynostosis, is locally abundant; but it is present at low levels in
Fgfr2 expression domains and at high levels in adjacent differentiated areas. Implantation of FGF2-soaked beads onto the fetal coronal suture
by ex utero surgery results in ectopic osteopontin expression, encircled by Fgfr2 expression, after 48 hours. It is suggested that
increased FGF/FGFR signaling in the developing skull, whether due to FGFR2 mutation or to ectopic FGF2, shifts the cell
proliferation/differentiation balance towards differentiation by enhancing the normal paracrine down-regulation of Fgfr2. In this model, a minor shift in the timing of the proliferation to diffentiation switch (due to loss of FGFR function or heightened levels of FGF) would favor differentiation and would result in the premature loss of the stem cell population and the induction of abnormal coronal suture (Iseki, 1997).
Fibroblast growth factor receptors (FGFRs) play major
roles in skeletogenesis, and activating mutations of the
human FGFR1, FGFR2 and FGFR3 genes cause
premature fusion of the skull bones (craniosynostosis). The patterns of expression of Fgfr1, Fgfr2
and Fgfr3 in the fetal mouse head, with specific reference
to their relationship to cell proliferation and differentiation
in the frontal and parietal bones and in the coronal suture, have been investigated.
Fgfr2 is expressed only in proliferating osteoprogenitor
cells; the onset of differentiation is preceded by down-regulation
of Fgfr2 and up-regulation of Fgfr1. Following
up-regulation of the differentiation marker osteopontin,
Fgfr1, osteonectin and alkaline phosphatase are down-regulated,
suggesting that they are involved in the
osteogenic differentiation process but not in maintaining
the differentiated state. Fgfr3 is expressed in the cranial
cartilage, including a plate of cartilage underlying the
coronal suture, as well as in osteogenic cells, suggesting a
dual role in skull development. Subcutaneous insertion of
FGF2-soaked beads onto the coronal suture on E15
resulted in up-regulation of osteopontin and Fgfr1 in the
sutural mesenchyme, down-regulation of Fgfr2, and
inhibition of cell proliferation. This pattern was observed
at 6 and 24 hours after bead insertion, corresponding to the
timing and duration of FGF2 diffusion from the beads. It is
suggested that (1) a gradient of FGF ligand, from high levels
in the differentiated region to low levels in the environment
of the osteogenic stem cells, modulates differential
expression of Fgfr1 and Fgfr2, and (2) signaling
through FGFR2 regulates stem cell proliferation whereas
signaling through FGFR1 regulates osteogenic
differentiation (Iseki, 1999).
FGF receptors and branching morphogenesis Branching morphogenesis of mouse submandibular glands is regulated by multiple growth factors. Ex vivo branching of intact
submandibular glands decreases when either FGFR2 expression is
downregulated or soluble recombinant FGFR2b competes out the endogenous growth factors. However, a combination of neutralizing antibodies to FGF1, FGF7 and
FGF10 is required to inhibit branching in the intact gland, suggesting that multiple FGF isoforms are required for branching. Exogenous FGFs added to submandibular epithelial rudiments cultured without mesenchyme induce distinct morphologies. FGF7 induces epithelial budding, whereas FGF10 induces duct
elongation, and both are inhibited by FGFR or ERK1/2 signaling inhibitors. However, a PI3-kinase inhibitor also decreases FGF7-mediated epithelial
budding, suggesting that multiple signaling pathways exist. FGF receptors were immunolocalized and changes in FGFR, FGF and MMP gene expression were analyzed to identify the mechanisms of FGF-mediated morphogenesis. FGFR1b and FGFR2b are present throughout the epithelium,
although FGFR1b is more highly expressed around the periphery of the buds and the duct tips. FGF7 signaling increases FGFR1b and FGF1 expression, and MMP2 activity, when compared with FGF10, resulting in increased cell proliferation and expansion of the epithelial bud, whereas
FGF10 stimulates localized proliferation at the tip of the duct. FGF7- and FGF10-mediated morphogenesis is inhibited by an MMP inhibitor and a neutralizing antibody to FGF1, suggesting that both FGF1 and MMPs are essential downstream mediators of epithelial morphogenesis. Taken together, these data suggests that FGFR2b signaling involves a regulatory network of
FGFR1b/FGF1/MMP2 expression that mediates budding
and duct elongation during branching morphogenesis (Steinberg, 2005).
FGF receptors and lung morphogenesis Mammalian lungs begin as an outpocket of the foregut, and depend on multiple stages of branching
morphogenesis and alveogenesis to reach their final form. An examination of fgf receptor gene expression
indicates that all four receptors (fgfr-1 to fgfr-4) are expressed in postnatal lungs at varying levels. Mice homozygous for a targeted mutation of fgfr-4 exhibit no overt abnormalities in the lungs
or any other organ. However, mice doubly homozygous for disruptions of the fgfr-3 and fgfr-4 genes
display novel phenotypes not present in either single mutant, which include pronounced dwarfism and lung
abnormalities. Lungs of fgfr-3(-/-);fgfr-4(-/-) animals, which are normal at birth, are completely blocked in
alveogenesis and do not form secondary septae to delimit alveoli. Consequently, air spaces in the lung are
expanded and no alveoli can be seen. The mutant lungs fail to downregulate postnatal elastin deposition
despite their normal levels of surfactant expression and cell proliferation. These data revealed a cooperative
function of FGFR-3 and FGFR-4 to promote the formation of alveoli during postnatal lung development (Weinstein, 1998).
FGF receptors and lung pancreatic development The development of the pancreas depends on epithelial-mesenchymal interactions. Fibroblast growth factors (FGFs) and their receptors (FGFRs 1-4) have been identified as mediators of epithelial-mesenchymal interactions in different organs. FGFR-2 IIIb and its ligands FGF-1, FGF-7, and FGF-10 are expressed throughout pancreatic development. In mesenchyme-free cultures of embryonic pancreatic epithelium, FGF-1, FGF-7, and FGF-10 stimulate the growth, morphogenesis, and cytodifferentiation of the exocrine cells of the pancreas. The role of FGFs signaling through FGFR-2 IIIb was further investigated by inhibiting FGFR-2 IIIb signaling in organocultures of pancreatic explants (epithelium + mesenchyme) by using either antisense FGFR-2 IIIb oligonucleotides or a soluble recombinant FGFR-2 IIIb protein. Abrogation of FGFR-2 IIIb signaling resulted in a considerable reduction in the size of the explants and in a 2-fold reduction of the development of the exocrine cells. These results demonstrate that FGFs signaling through FGFR-2 IIIb play an important role in the development of the exocrine pancreas (Miralles, 1999).
FGF receptors and epidermal development To understand the role Fgf signalling in skin and hair follicle
development, the phenotype was analyzed of mice deficient for Fgfr2-IIIb and
its main ligand Fgf10. These studies show that the severe epidermal
hypoplasia found in mice null for Fgfr2-IIIb is caused by a lack of the basal cell proliferation that normally results in a stratified epidermis. Although at term the epidermis of Fgfr2-IIIb null mice is only two to three cells thick, it expresses the classical markers of epidermal differentiation and establishes a functional barrier. Mice deficient for Fgf10 display a similar but less severe epidermal hypoplasia. By contrast,
Fgfr2-IIIb–/– (but not
Fgf10–/–) mice produce significantly fewer
hair follicles, and their follicles were developmentally retarded. Following
transplantation onto nude mice, grafts of
Fgfr2-IIIb–/– skin show impaired hair
formation, with a decrease in hair density and the production of abnormal
pelage hairs. Expression of Lef1, Shh and Bmp4 in
the developing hair follicles of Fgfr2-IIIb–/–
mice is similar to wild type. These results suggest that Fgf signalling
positively regulates the number of keratinocytes needed to form a normal
stratified epidermis and to initiate hair placode formation. In addition, Fgf signals are required for the growth and patterning of pelage hairs (Petiot, 2003).
Loss of Fgf9 has been shown to result in a block of testis development and a male to female sex-reversed phenotype; however, the function of Fgf9 in sex determination was unknown. Fgf9 is now shown to be necessary for two steps of testis development just downstream of the male sex-determining gene, Sry: (1) for the proliferation of a population of cells that give rise to Sertoli progenitors; and (2) for the nuclear localization of an FGF receptor (FGFR2) in Sertoli cell precursors. The nuclear localization of FGFR2 coincides with the initiation of Sry expression and the nuclear localization of SOX9 during the early differentiation of Sertoli cells and the determination of male fate (Schmahl, 2004).
It has been known for some time that many cell-surface growth factor receptors can accumulate within the nucleus. However, the biological relevance of this event is not known. It has been speculated that nuclear growth factor receptors may act as weak transcription factors, topoisomerases and/or nuclear kinases. Nuclear FGF receptors have been observed in spliceosomes. This is particularly interesting, since several components of the sex-determination pathway (including SRY, SOX9 and the +KTS isoform of WT) have been shown to associate with splicing factors, and have been demonstrated to have splicing activity. Another function of nuclear FGF receptors may be to phosphorylate nuclear substrates; forced nuclear translocation of FGF receptors leads to an increase in the phosphorylation of nuclear proteins, and some activities of the nuclear receptor are abolished by deactivation of the kinase domain. The discovery of the sex-specific subcellular localization of FGFR2 in the nuclei of Sertoli precursors provides a well-characterized biological context in which to study the function of nuclear growth factor receptors. In this context, the transition of FGFR2 from the cell membrane to the nucleus suggests that the nuclear localization of cell-surface receptors is linked to the initiation of cell differentiation. It is not yet clear how proliferation of Sertoli cell precursors in the coelomic epithelium and subsequent commitment to the Sertoli fate are interwoven; however, these findings suggest that FGF signaling may be involved in bridging these two processes essential to testis development (Schmahl, 2004).
The fibroblast growth factor (FGF) family consists of 22 members and
regulates a broad spectrum of biological activities by activating diverse
isotypes of FGF receptor tyrosine kinases (FGFRs). Among the FGFs, FGF7 and
FGF10 have been implicated in the regulation of prostate development and
prostate tissue homeostasis by signaling through the FGFR2 isoform. Using
conditional gene ablation with the Cre-LoxP system in mice, a
tissue-specific requirement has been demonstrate for FGFR2 in urogenital epithelial cells - the
precursors of prostatic epithelial cells - for prostatic branching
morphogenesis and prostatic growth. Most Fgfr2 conditional null
(Fgfr2cn) embryos developed only two dorsal prostatic (dp)
and two lateral prostatic (lp) lobes. This contrasts to wild-type prostate,
which has two anterior prostatic (ap), two dp, two lp and two ventral
prostatic (vp) lobes. Unlike wild-type prostates, which are composed of well
developed epithelial ductal networks, the Fgfr2cn
prostates, despite retaining a compartmented tissue structure, exhibited a
primitive epithelial architecture. Moreover, although
Fgfr2cn prostates continued to produce secretory proteins
in an androgen-dependent manner, they responded poorly to androgen with
respect to tissue homeostasis. The results demonstrate that FGFR2 is important
for prostate organogenesis and for the prostate to develop into a strictly
androgen-dependent organ with respect to tissue homeostasis but not to the
secretory function, implying that androgens may regulate tissue homeostasis
and tissue function differently. Therefore, Fgfr2cn
prostates provide a useful animal model for scrutinizing molecular mechanisms
by which androgens regulate prostate growth, homeostasis and function, and may
yield clues as to how advanced-tumor prostate cells escape strict androgen
regulations (Lin, 2007).
The mouse seminal vesicle shape (svs) mutation is a spontaneous recessive
mutation that causes branching morphogenesis defects in the prostate gland and
seminal vesicles. Unlike many other mutations that reduce prostatic and/or
seminal vesicle branching, the svs mutation dramatically reduces branching
without reducing organ growth. Using a positional cloning approach, the svs mutant lesion was identified as a 491 bp insertion in the tenth intron of
Fgfr2 that results in changes in the pattern of Fgfr2
alternative splicing. An engineered null allele of Fgfr2 failed to
complement the svs mutation proving that a partial loss of FGFR2(IIIb)
isoforms causes svs phenotypes. Thus, the svs mutation represents a new type
of adult viable Fgfr2 allele that can be used to elucidate receptor
function during normal development and in the adult. In the developing seminal
vesicles, sustained activation of ERK1/2 was associated with branching
morphogenesis and this was absent in svs mutant seminal vesicles. This defect
appears to be the immediate downstream effect of partial loss of FGFR2(IIIb)
because activation of FGFR2(IIIb) by FGF10 rapidly induced ERK1/2 activation,
and inhibition of ERK1/2 activation blocked seminal vesicle branching
morphogenesis. Partial loss of FGFR2(IIIb) was also associated with
down-regulation of several branching morphogenesis regulators including
Shh, Ptch1, Gli1, Gli2, Bmp4, and Bmp7. Together with
previous studies, these data suggest that peak levels of FGFR2(IIIb) signaling
are required to induce branching and sustain ERK1/2 activation, whereas reduced levels support ductal outgrowth in the prostate gland and seminal vesicles (Kuslak, 2007).
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