Functions and Regulations of Fibroblast Growth Factor Signaling During

8/10/2019 Functions and Regulations of Fibroblast Growth Factor Signaling During

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Functions and regulations of fibroblast growth factor signaling during

embryonic development

Bernard Thisse, Christine Thisse *

Institut de Genetique et de Biologie Moleculaire et Cellulaire, UMR 7104, CNRS/INSERM/ULP, 1 rue Laurent Fries, BP 10142, CU de Strasbourg,

67404 ILLKIRCH cedex, France

Received for publication 16 June 2005, revised 29 July 2005, accepted 5 September 2005

Available online 10 October 2005

Abstract

Fibroblast growth factors (FGF) are secreted molecules which function through the activation of specific tyrosine kinases receptors, the FGF

receptors that transduce the signal by activating different pathways including the Ras/MAP kinase and the phospholipase-C gamma pathways.

FGFs are involved in the regulation of many developmental processes including patterning, morphogenesis, differentiation, cell proliferation or

migration. Such a diverse set of activities requires a tight control of the transduction signal which is achieved through the induction of different

feedback inhibitors such as the Sproutys, Sef and MAP kinase phosphatase 3 which are responsible for the attenuation of FGF signals, limiting

FGF activities in time and space.

D 2005 Elsevier Inc. All rights reserved.

Keywords: FGF; FGF receptors; Ras/MAP kinase; HSPG; Sprouty; Sef; MKP3

Introduction

Fibroblast growth factors represent a large family of secreted

molecules. Upon binding to their cognate receptors, FGFs

activate signal transduction pathways which are required for

multiple developmental processes. The wide-ranging biological

roles of FGFs, the variety of signaling pathways activated and

the complex and dynamic expressions of FGF ligands and

receptors imply that FGF signaling must be tightly regulated.

The scope of the present review is to provide readers with

updated information about FGF signaling. After a short

introduction on FGFs evolutionary history and structural

characteristics and on FGF receptors (FGFRs) particularities,various functions of the FGF signaling will be presented,

focusing on their implication in development. The last part of

the review will describe the attenuation of FGF signaling mainly

dependent on the Ras/MAP kinase signaling pathway, through

the action of different factors, such as the Sproutys, Sef or MAP

kinase phosphatase 3. Recent publication of various studies has

led to controversial results and we will present the raising

debate on how FGF can be regulated and on how multiple

feedback regulators of the FGF pathway act at different levels ofthe signal transduction cascades to attenuate FGF signaling.

Key structural properties of FGFs and FGF Receptors

FGFs represent an extended family of polypeptide growth

factors found through evolution, ranging from nematode to

human whereas they have not been identified in unicellular

organisms (for review,Itoh and Ornitz, 2004). Ininvertebrates,

only three Drosophila genes (branchless,pyramus and thisbe)

have been found and two (egl-17 and let-756) in Caenorhab-

ditis elegans. In contrast, in vertebrates, a large number ofFGF

genes has been identified: 10 FGFs in zebrafish (FGF2 4, 6,8,10 ,17a,17b,18,24), 6 inXenopus(FGF2 4,810), 13 in

chicken (FGF1 4, 810, 12, 13, 16, 1820), 22 genes in

mouse (FGF1 18, 2023) and human (FGF1 14, 1623).

FGFgenes are scattered throughout the genome, indicating

that the FGF gene family was generated both by gene and

chromosomal duplication and translocation during evolution

(for reviewOrnitz and Itoh, 2001).

All FGFs share an internal core of similarity as well as a

high affinity for heparin. FGFs act through binding and

activation of FGFRs which are tyrosine kinase receptors that

contain three immunoglobulin-like domains and a heparin-

0012-1606/$ - see front matterD

2005 Elsevier Inc. All rights reserved.doi:10.1016/j.ydbio.2005.09.011

* Corresponding author.

E-mail address: [email protected] (C. Thisse).

Developmental Biology 287 (2005) 390 402

www.elsevier.com/locate/ydbio


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binding sequence (Lee et al., 1989; Johnson et al., 1990,Fig.

1). FGFR forms are expressed in two possible ways: by the

expression of splice variants of a given FGFR gene or by the

expression of different FGFR genes (Johnson and Williams,

1993). Alternative splicing specifies the sequence of the

carboxy-terminal half of the Ig domain III, resulting in either

IIIb or IIIc isoforms (Miki et al., 1992; Chellaiah et al., 1994).

This alternative splicing event is regulated in a tissue-specific

manner and dramatically affects ligand receptor binding

specificity (seePowers et al., 2000).

Fig. 1. FGF receptors and FGF signal transduction. FGFRs are modular proteins comprising 3 immunoglobulin domains (IgI, IgII and IgIII). IgI and IgII are

separated by an acidic box (AD). IgII contains a heparin binding domain (HBD). The IgIII domain is followed by a unique transmembrane (TM), a juxtamembrane

(JM) and a kinase domain (KD) interrupted by an interkinase domain (IKD). FGF ligands linked to heparin sulfate proteoglycan (HSPG) bind to IgII and IgIII of

FGFR. This results in the dimerization and the subsequent transactivation by phosphorylation of specific tyrosine residues. The main two transduction pathways

involve the phospholipase C-g(PLCg) and the Ras/MAP kinase. The SH2 domain of the PLCginteracts with the phosphorylated Y766 of the activated receptor. The

activated PLCg hydrolyzes the phosphatidyl-inositol-4,5-diphosphate (PIP2) to inositol-1,4,5-triphophate (IP3) and the diacylglycerol (DAG). IP3 releases Ca 2+

while DAG activates the protein kinase C-y (PKCy). Activated PKCy activates Raf by phosphorylating its S338 and stimulates the downstream pathway in a Ras

independent manner. The main pathway involves the interaction of the docking protein FRS2awith the amino-acid residues 407 433 (Xu et al., 1998). This protein

is activated by phosphorylation on multiple tyrosine residues and subsequently interacts and activates Grb2 linked to Sos, a nucleotide exchange factor involved in

the activation of Ras. Activated Ras then activates Raf which stimulates MEK which in turn phosphorylates the MAP kinase ERK. This last activated component

translocates to the nucleus and phosphorylates specific transcription factors of the Ets family which in turn activate expression of specific FGF target genes. P:phosphorylation.

B. Thisse, C. Thisse / Developmental Biology 287 (2005) 390 402 391


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Role of Heparin Sulfate Proteoglycans in FGF ligand

binding

In addition to high affinity for their receptors, members of

the FGF family have strong affinity for heparin (Burgess and

Maciag, 1989). FGF FGFR interaction and signaling are

further regulated by the spatial and temporal expression ofendogenous heparan sulfate proteoglycan (HSPG). HSPGs are

cell-surface and extracellular matrix macromolecules that

comprise a core protein to which heparan sulfate (HS)

glycosaminoglycan (GAG) chains are attached. HSPGs play

crucial roles in regulating key developmental pathways such as

the FGF signaling (for review, Lin, 2004). The genetic

evidences of such a role for HSPGs come from the isolation

of mutants, both in invertebrates and vertebrates that show

defective FGF signaling (Lin et al., 1999; Nybakken and

Perrimon, 2002). Biochemical (Nakato and Kimata, 2002) and

cristallographic studies (Schlessinger et al., 2000; Pellegrini et

al., 2000) have revealed the importance of 60 sulfation of HSfor FGF signaling which appears to be required for FGF1

FGFR2 and FGF2FGFR1 interactions. HSPGs were found to

directly interact with FGFs and their receptors in a ternary

complex on the cell surface (Ornitz, 2000; Pellegrini, 2001).

One possibility could be that Heparin/HSPGs within the FGF

signaling complex facilitate the FGFFGFR interaction. The

second possibility could be that Heparin/HSPGs enhance the

stability of a FGF FGFR complex. Examination of the

spatiotemporal changes in HS to promote complexes formation

with FGFRs and FGFs led to the finding that these changes

depend on glycosyltransferases and sugar sulfotransferases.

Furthermore, for each FGF FGFR pair examined, unique

developmental HS binding patterns were identified that

correlate with different HS binding requirements, as well as

variations in FGF signaling. These results suggested that each

FGFFGFR combination seeks distinct HS domains that are

spatially and temporally regulated during development. These

domains are unique to HS and distinguish it from heparin,

which is highly sulfated and lacks any domain structure,

explaining the ability of these FGFFGFR pairs to interact

equally with heparin in vitro. Importantly, the HS activity

necessary for ternary complex assembly does not represent the

additive binding requirements of individual FGFs or FGFRs;

rather, it represents requirements dictated by the synergistic

interaction of the FGFs, HS and FGFRs. Finally, given that HSspecifically mediates each FGFFGFR interaction examined,

these results suggest that developmental changes in HS may

also specifically modulate the signaling of other families of

morphogens and growth factors (Allen and Rapraeger, 2003).

In addition, these interactions limit FGF diffusion and release

into interstitial spaces, regulating FGF activity in a given tissue

(Moscatelli, 1987; Flauennhaft et al., 1990).

FGF transduction pathway

FGFR transmits extracellular signals to various cytoplasmic

signal transduction pathways through tyrosine phosphorylation

(Fig. 1). FGFRs exist as inactivated monomers, which are

activated when two FGF molecules connected by a heparan

sulfate proteoglycan bind to the extracellular IgII and IgIII

domains of the receptor leading to its homodimerization

(Schlessinger et al., 2000). This dimerization brings together

the intracellular domain of the receptor leading to transauto-

phosphorylation of several critical tyrosine residues. Activation

of the receptor allows proteins containing Src homology (SH2)or phosphotyrosine binding (PTB) domains to bind to sequence

recognition motifs in FGFR1, resulting in phosphorylation and

activation of these proteins (Pawson, 1995; Forman-Kay and

Pawson, 1999; Dhalluin et al., 2000). For example, the

Phospholipase C-g (PLCg) was identified to be associated

with FGFR following ligand-dependent activation. This asso-

ciation is due to binding between the SH2 domain of PLCgand

Tyr 766 of FGFR1. Activated PLCg can hydrolyze phospha-

tidylinositol-4,5-diphosphate (PIP2) to inositol-1, 4, 5-triphos-

phate (IP3) and diacylglycerol (DAG). IP3 induces Ca2+

release from intracellular stores, whereas DAG is a protein

kinase C activator. The activated protein kinase C-y (PKCy) isable to phosphorylate and to stimulate Raf therefore leading to

activate the MAP kinase pathway in a Ras-independent manner

(Ueda et al., 1996).

The main signaling activated through the stimulation of

FGFRs is the Ras/MAP kinase pathway. Links between

elements of this pathway and the activated FGFR involve the

interaction of the juxtamembrane domain of FGFR with the

FGFR substrate 2a (FRS2a) which is a membrane-anchored

docking protein. FRS2 lacks an SH2 domain, but contains a

phosphotyrosine binding domain (Kouhara et al., 1997), which

was shown to mediate phosphotyrosine-independent interac-

tion with amino-acid residues 407433 in FGFR1. Activation

of FGFR1 leads to tyrosine phosphorylation of FRS2 at several

sites. This allows the binding of Grb2, a small adaptor

molecule, which exists in complex with the nucleotide

exchange factor Sos and is involved in the activation of the

GTP-binding protein Ras. In this complex, Sos catalyzes the

exchange of GDP for GTP on Ras, promoting Ras activation as

well as downstream elements of the pathway, comprising Raf,

MEK and MAP kinases (ERK1, 2), the last member of which

finally enters the nucleus and phosphorylates target transcrip-

tion factors (Sternberg and Alberola-Ila, 1998). Among these

are the Ets domain containing factors ERM and PEA3

(Wasylyk et al., 1998; Sharrocks, 2001). They belong to a

family of transcription factors, which share a winged-helixloophelix domain with which they bind to DNA as

monomers. They are able to form heterocomplexes with

transcription factors and are activated most prominently by

the MAP kinase(Wasylyk et al., 1998).

A study performed during the course of somite development

revealed the contribution of these Ets-domain containing

proteins activated by FGFs. The FGF signal secreted from

the myotome induces formation of a scleraxis (Scx)-expressing

tendon progenitor population in the sclerotome. Brent and

Tabin (2004) showed that transcriptional activation by Pea3

and Erm in response to FGF signaling is both necessary and

sufficient for Scx expression in the somite. They proposed that

the domain of the somitic tendon progenitors is regulated both

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by the restricted expression of Pea3 and Erm, and by the

precise spatial relationship between these Ets transcription

factors and the FGF signal originating in the myotome.

Implication of FGF signaling during development

FGFs were described to be implicated in multiple roles suchas patterning, morphogenesis, differentiation, regulation of cell

proliferation or migration (for review see Goldfarb, 1996;

Powers et al., 2000; Coumoul and Deng, 2003). In the next

paragraphs, we will illustrate major functions of FGF signaling

during development.

Early patterning and dorso-ventral axis formation

Induction and patterning of mesoderm is one of the earliest

events in which FGF signaling was revealed to be essential.

The first mesodermal inducer identified was basic FGF (Slack

et al., 1987), for which its role is evolutionarily conserved.The effect of FGF on mesoderm formation was initially

studied in Xenopus in which not only FGF was described to

have a mesoderm inducing capacity (Slack et al., 1987;

Kimelman and Kirschner, 1987; Kimelman et al., 1988) but

components of the FGF signaling pathway such as FGFRs,

HSPGs, FRS2, Grb2 and the Ras downstream cascade, when

inhibited, were shown to block mesoderm formation and to

induce both gastrulation and posterior defects (Whitman and

Melton, 1992; MacNicol et al., 1993; Umbhauer et al., 1995;

LaBonne, 1995; Gotoh, 1995).

Involvement of FGFs in the establishment of the dorso-

ventral (D/V) axis has been extensively studied using the

zebrafish model. Genetic analysis revealed that BMP2b/BMP7

heterodimers (Schmid et al., 2000)act as morphogens secreted

ventrally and are responsible for the specification of ventral cell

fates. The expression of BMPgenes, initially activated in the

whole blastula becomes progressively restricted ventrally. This

ventral restriction coincides with the spreading of FGF activity

from the dorsal side of the embryo, suggesting an implication

of FGF signaling in the dorsal downregulation of BMP gene

expression. Consistently, general activation of the FGF/Ras/

MAP kinase signaling pathway inhibits BMP gene expression

in the whole blastula. Conversely, inhibition of FGF signaling

causes BMPgene expression to enlarge dorsally, leading to an

expansion of ventral cell fates. Altogether, this shows that FGFsignaling is essential to delimit the expression domain ofBMPs

in blastula stage embryos. Therefore, FGFs act upstream of the

ventral morphogens and function as initial signals for the

establishment of the D/V patterning (Furthauer et al., 2004).

FGF signaling was also identified to be implicated in

ventro-posterior mesodermal tissue maintenance. Consistently,

blocking FGF signaling by dominant-negative FGFR results in

deletions of tail and trunk structures (Amaya et al., 1991;

Griffin et al., 1995). As well, targeted disruption of mouse

FGFR1 leads to the disorganization of axial mesoderm and to a

defective development of the posterior structures. FGFs control

the specification and maintenance of mesoderm by regulation

of T box genes (Griffin et al., 1995; Smith et al., 1991; Strong

et al., 2000; Sun et al., 1999; Ciruna and Rossant, 2001; Griffin

et al., 1998; Zhao et al., 2003). One FGF mesodermal target is

brachyury, required for posteriormesoderm and axis formation

in mouse, zebrafish andXenopus(Smith et al., 1991; Herrmann

et al., 1990; Halpern et al., 1993; Conlon et al., 1996). In

Xenopus, Brachyury induces eFGF, which is required for

myogenic celllineage (Stanley et al., 2001; Fisher et al., 2002)and vice versa (Isaacs et al., 1994; Schulte-Merker and Smith,

1995).

Role of FGFs on cell movements

Evidence has been provided that FGF signaling plays a

direct role in cell movements during convergence extension

(Nutt et al., 2001). In the mouse, FGF signaling may affect

gastrulation movements via the transcription factor snail

(Ciruna and Rossant, 2001). FGFR1 mutant embryos die at

late gastrulation, showing defects in cell migration, cell fate

specification and patterning. FGFR1 mutant cells may fail tomigrate properly because they maintain high levels of E-

cadherin resulting from a snail downregulation. An additional

study performed in the mouse revealed that, in the absence of

both FGF8 and FGF4, epiblast cells move into the streak and

undergo an epithelial-to-mesenchymal transition; however,

most cells then fail to move away from the streak. As a

consequence, no embryonic mesoderm- or endoderm-derived

tissues develop demonstrating that signaling via FGF8 and/or

FGF4 is required for cell migration away from the primitive

streak(Sun et al., 1999).

Moreover, there is an increasing evidence for a direct

chemotactic response of migrating cells in response to FGF

signals. As such, FGF2 and FGF8 are potent chemoattractants

in migration of mesencephalic neural crest cells (Kubota and

Itoh, 2000) and FGF2 and FGF4 attract mesenchymal cells

during limb bud development in the mouse(Webb et al., 1997;

Li and Muneoka, 1999). Also, FGF10 exerts a potent

chemoattractive effect to direct lung distal epithelial buds to

their destination(Park et al., 1998). Furthermore, FGFs can act

as chemotactic signals to coordinate cell movements during

gastrulation. In the chick, cells can be guided by environmental

signals and beads coated with FGFs alter their trajectories. The

observed movement patterns of anterior streak cells can be

explained by an FGF8-mediated chemorepulsion of cells away

from the streak followed by chemoattraction towards an FGF4signal produced by the forming notochord(Yang et al., 2002).

Neural induction and FGF signaling

Several studies on neural induction have led to the ?default

modelX which proposes that ectodermal cells are fated to

become neural by default. In this model, cells are normally

inhibited from a neural fate by BMPs that are expressed in the

ectoderm, from which they must be released for neural

induction to occur (for reviews, Hemmati-Brivanlou and

Melton, 1997; Weinstein and Hemmati-Brivanlou, 1999).

Additional data have suggested that FGF signaling plays a

crucial role for an early step of neural induction in the chick. In



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this specie, induction of neural tissue precedes the formation of

the Hensens node. FGFs are sufficient to induce preneural

markers and inhibition of FGF signaling blocks neural

induction by Hensens node (Streit et al., 2000; Wilson et al.,

2000; Alvarez et al., 1998; Storey et al., 1998). FGF signals

repress BMP expression which leads to induction of neural

tissue. On the contrary, when FGFs are inhibited, BMPexpression is maintained and neural fate is blocked (Wilson

et al., 2000). FGFs may block BMP signaling during neural

induction of the chick by inducing Churchill which is required

for the expression of Smad-interacting protein 1 (Sip1) in the

neural plate. Sip1 binds and represses Smad1/5 blocking

therefore BMP signaling (Sheng et al., 2003).

In Xenopus, the role of FGF signaling in neural induction

has also been intensively studied. However, its implication in

neural induction has led to conflicting results. Several

investigators suggested that FGF signaling is required directly

for neural induction (Launay et al., 1996; Hongo et al., 1999;

Streit et al., 2000; Bertrand et al., 2003). In most cases,however, experiments were performed in a background where

the BMP signaling was partially attenuated (Kengaku and

Okamoto, 1995; Lamb and Harland, 1995; Ribisi et al., 2000).

In addition, several studies using dominant negative FGFR1

have led to different results showing that neural tissue forms

even when FGF signaling is inhibited (Kroll and Amaya, 1996;

Ribisi et al., 2000; Barnett et al., 1998; McGrew et al., 1997;

Holowacz and Sokol, 1999). It appears that FGF signaling

required for anterior neural development may be mediated

through FGFRs other than FGFR1, such as through FGFR4a

(Hongo et al., 1999).

In contrast, other studies report a requirement for FGFs in

the antero-posterior patterning rather than the initial induction

of the neural plate(Holowacz and Sokol, 1999). FGFs are able

to change anterior neural fate to more posterior neural cell

types (Umbhauer et al., 2000; Lamb and Harland, 1995; Ribisi

et al., 2000; Holowacz and Sokol, 1999; Cox and Hemmati-

Brivanlou, 1995). This may thus involve interactions with the

Wnt signaling pathway (McGrew et al., 1997; Kazanskaya et

al., 2000; Domingos et al., 2001; Kudoh et al., 2002).

Finally, a controversial study provides evidence that BMP

inhibition is required in the chick, but only as a relatively late

step. BMP inhibition would not be sufficient to cause

competent cells to acquire neural fates either in chick or in

Xenopus. In the frog, FGF synergizes with BMP inhibition toinduce neural markers. In chick, inhibition of BMP signaling

with Wnt antagonists and/or FGF is not sufficient for neural

induction. Neural induction would not occur by default but

would rather involve a succession of signaling events with

some players which would remain to be identified(Linkerand

Stern, 2004).

Midbrain hindbrain patterning

Early patterning of the vertebrate presumptive midbrain and

rhombomere 1 (rh1) is regulated by a local organizer located at

the mid/hindbrain junction(Liu and Joyner, 2001), a territory

expressing FGF8. This factor has organizer properties and is

required for midbrain and cerebellum development(Meyers et

al., 1998, Reifers et al., 1998). FGF17 and FGF18 are also

expressed in the mid/hindbrain junction in broader domains

than FGF8, including posterior midbrain(Maruoka et al., 1998;

Xu et al., 1998). Loss-of-function of FGF17 in the mouse

results in truncation of the posterior midbrain and reduced

proliferation of the anterior cerebellum. Removal of one copyof fgf8 in a fgf17 mutant background leads to exaggerated

cerebellum phenotype(Xu et al., 2000). To complicate matters,

fgf8 is differentially spliced to generate FGF8a and FGF8b

isoforms. FGF8a misexpression causes expansion of the

midbrain and FGF8b transforms the midbrain into cerebellum.

The initial effect of FGF8b is to reduce growth of the midbrain.

Therefore, FGF8a and FGF8b have distinct activities, both on

growth and regulation of gene expression (reviewed in Liu and

Joyner, 2001; Sato et al., 2001).

Interactions of FGF8a and FGF8b with FGF17 and FGF18

have been studied. It appears that FGF8b after being induced in

the presumptive rh1 territory induces FGF18 in surroundingtissue, further extending the gradient of FGF RNA expression.

FGF8b maintains two negative feedback loops by inducing the

expression of the negative feedback FGF inhibitors Spry1 and

Spry2 and repressing FGFR2 and FGFR3 (Liu et al., 2003).

FGF17, FGF18 proteins and possibly FGF8a as well as a low

level of FGF8b then regulate proliferation of the midbrain and

cerebellum(Liu et al., 2003; Chi et al., 2003).

Limb induction and morphogenesis

The role of FGFs has been established in induction,

initiation and maintenance of limb development (reviewed in

Martin, 1998). Limb bud initiation is triggered by FGF8

inducing the expression of FGF10. FGF10 then in turn induces

FGF8 in ectodermal cells resulting in the formation of the

apical ectodermal ridge (AER). FGFs from the AER maintain

cell proliferation in the progress zone (a population of

undifferentiated mesenchymal cells located near the distal tip

of the limb bud, adjacent to the AER). FGF2 (Fallon et al.,

1994), FGF4 (Laufer et al., 1994)and FGF8(Crossley etal.,

1996) induce sonic hedgehog (shh) expression in the zone of

polarizing activity (ZPA). Outgrowth and patterning of the limb

result from the combined effects of FGF and Shh and

regulation of various genes, including members of the Hox

family (reviewed inMartin, 1998).A first model proposed for explaining the patterning of the

limb development along the proximaldistal (PD) axis is the

progress zone model(Summerbell et al., 1973) which explains

that as the limb grows, cells are pushed out of the progress zone

and become fixed in the positional value that they acquired.

Cells that leave the proximal zone early will adopt a proximal

fate and cells that leave the progress zone late will form distal

elements (Sauders, reviewed in Niswander, 2003). The

conclusion from this model is that the PD fate is specified

progressively. Recent studies have provided an alternative

model on how the AER may influence the PD pattern. This

second model proposes that all PD fates are already specified

within the early limb bud rather than progressively (Dudley et



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al., 2002). In the absence of FGF8 in mice, the proximal

element of the limb is reduced or even lost. However, the distal

elements are formed quite normally because other FGFs are

activated (Lewandoski et al., 2000; Moon and Capecchi, 2000).

These results of loss of proximal but not of distal elements

cannot be interpreted within the progress zone model. In

addition, new experimental data have proposed that FGFsfunction is first to influence the initial size of the limb (Sun et

al., 2002). In FGF8 mutant mice, early limb bud is smaller.

This phenotype is detected right after FGF8 expression is

detectable, excluding the possibility that FGF8 alters cell

proliferation or cell death. Instead, FGF8 would (by affecting

morphogenetic movements or cell adhesion) influence the

number of cells in the limb bud by preventing their death. In

mutant limbs, death of proximal but not distal mesenchyme can

be observed. The PD precartilage condensations are formed but

smaller. Sun et al. (2002)proposed that the AER generates an

adequate number of mesenchymal cells for correctly sized

condensations to form. When the AER is disrupted, the numberof skeletal elements is decreased resulting in smaller or in

absence of condensations arguing that the FGFs function is to

control mesenchymal cell number but not the PD patterning.

Bone formation

The importance of FGF signaling in skeletal development

was first revealed with the discovery that a point mutation in

the transmembrane domain of FGFR3 is the etiology of

achondroplasia, the most common genetic form of dwarfism

in human (Rousseau et al., 1994; Shiang et al., 1994). A major

role of FGFs and FGFRs has then been scored by finding

missense mutations resulting in more than 15 human disorders,

ranging from skeletal dysplasias, craniosysnostosis syndromes

or short stature (for reviewCoumoul and Deng, 2003). In the

bone, FGF2 and FGF9 transcripts are found in mesenchymal

cells and osteoblats (Gonzalez et al., 1996; Kim et al., 1998).

FGF18 is expressed in mesenchymal cells, in differentiating

osteoblasts during calvarial bone development and in the

perichondrium of long bones (Ohbayashi et al., 2002). The

action of FGFs is dependent on the spatiotemporal pattern of

expression of FGFRs like FGFR1 and FGFR2 which are

located in mesenchymal cells during condensation of mesen-

chyme prior to deposition of bone matrix at early stages of long

bone development and in the cranial sutures (Orr-Urtreger etal., 1991; Delezoide et al., 1998). Later, they are found in

preosteoblasts and osteoblasts together with FGFR3 (Molteni

et al., 1999). Overexpression of FGF2 in mouse induces

abnormal bone formation (Coffin et al., 1995)whereas its loss-

of-function leads to inhibition of bone formation (Monteroet

al., 2000). Mice lacking FGF18 display ossification and cranial

sutures closure delay (Liu et al., 2002; Ohbayashi et al., 2002).

Altogether, FGF signaling appears to regulate cell proliferation

and differentiation positively in membranous and long bone

osteogenesis. FGF2 was shown also to act in vivo on osteoblast

precursor cells replication to increase osteoblast number. FGF

signaling also controls genes involved in osteoblast apoptosis.

High FGF signaling may reduce apoptosis of immature

osteoblasts and thereby enhance the osteoblast population,

whereas continuous signaling may promote apoptosis in more

mature osteoblasts, therefore limit the early increase in the

osteoblasts pool. FGFs interact with other growth factors

signaling pathways to regulate osteoblast function. For

example, FGF2 and FGFR2 inhibit the expression of the

BMP antagonist Noggin in the cranial sutures, resulting inincreased BMP4 activity and suture fusion (Warren et al.,

2003). Therefore, FGF signaling can control cranial suture

fusion indirectly through BMP signaling. FGFs regulate also

genes involved in matrix degradation(Kawaguchi et al., 1995)

and induce the expression of tissue inhibitors of metallopro-

teinases (Delany and Canalis, 1998; Varghese et al., 1995)

indicating that FGFs (mainly FGF2) may modulate bone matrix

proteolysis by regulating collagenase activity. Overall, activa-

tion of FGF signaling isable to regulate genes involved at all

steps of osteogenesis (Fig. 2). This array of genes may

participate in the regulation of cell progression from osteopro-

genitor cells to the end of the osteoblast life (for review, Marie,2003). Further studies have led to the identification of signaling

pathways that are involved in FGF signaling in osteoblasts,

mainly MAP kinase signal transduction cascade(Mansukhani

et al., 2000; Debiais et al., 2001).

Attenuation of FGF signaling

The multiple range of biological effects of FGFs and the

variety of signaling pathways activated by this family of

ligands imply that FGF signaling must be tightly regulated

regarding timing, duration and spread of the signal. This can be

achieved by both positive and negative feedback loops. FGF

signaling can be regulated at many levels in the extracelular

space and within the cell. A growing number of proteins have

been identified that specifically regulate the activity of FGF

signaling pathways through FGFRs. Many of these regulatory

factors belong to the FGF synexpression group (Furthauer et

al., 2002; Niehrs and Meinhardt, 2002; Tsang et al., 2002). Not

only are these factors coexpressed with FGFs but they are

themselves regulated by FGF signaling and inhibit FGF

signaling by establishing a negative feedback loop.

The Sprouty proteins

The first feedback regulator of the FGF pathway isolated isSprouty (Spry). Spry was originally identified as an antagonist

of FGF signaling that patterns apical branching in Drosophila

tracheae (Hacohen et al., 1998). In spry mutants, ectopic

airway branches are induced due to excess of FGF signaling.

Spry was then described to be a general inhibitor of receptor

tyrosine kinase (RTK) signaling (for review,Kim and Bar-Sagi,

2004). Spry proteins are evolutionarily conserved with four

members (Spry1, 2, 3, 4) identified in mammals (Dikic and

Giordano, 2003). These proteins share a conserved carboxy-

terminal cystein-rich domain, necessary for their specific

localization and function. Their amino-terminal part is diver-

gent except that they possess an invariant tyrosine phospho-

rylation site. It is thought that this sequence divergence dictates



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various functions by mediating different protein protein

interactions. Gain- and/or loss-of-functions studies in the

mouse, Xenopus and zebrafish (Minowada et al., 1999;

Mailleux et al., 2001; Nutt et al., 2001; Furthauer et al.,

2001, 2004) have demonstrated that Sprys antagonize FGF

signaling.Spryexpression is induced by the FGF signaling and

has various biological consequences, including regulation of

cell proliferation and differentiation, endocytic sorting of

activated RTKs, control of cytoplasmic calcium concentration,

migratory behavior of cells, retro-control of D/V patterning and

lung and bone development. Spry genes contribute also to

reciprocal epithelial mesenchymal and stromal signaling

controlling ureteric branching, which involves the coordination

of Fgf/Wnt11/Gdnf pathways during mammalian kidney

development(Chi et al., 2004).

Study of Sprys function has led to intense investigation and

controversy(Fig. 3). Works from many groups have identified

Sprys as negative regulators of the MAP kinase ERK.

However, contradictory reports have identified Spry inhibitionupstream of Ras(Casci et al., 1999; Hanafusa et al., 2002) or at

the level of Raf(Reich et al., 1999; Sasaki et al., 2003; Yusoff

et al., 2002). Moreover, Spry family members have varying

interacting partners, such as c-Cbl, Grb2, Raf1 and FRS2 (for

review, Dikic and Giordano, 2003).

The mechanisms by which Sprys interfere with ERK

activation have been extensively studied on EGF signaling

pathway and have come from the finding that Spry proteins are

regulated by tyrosine phosphorylation (Tyr55 in spry2, Tyr53

in Spry1/4) in response to stimuli. Phosphorylated Tyr55 serves

as a docking site for the SH2 domain of c-Cbl (Egan et al.,

2002; Wong et al., 2002; Rubin et al., 2003; Hall et al., 2003 ).

Usually, c-Cbl binds to phosphorylated tyrosines on the

activated EGFR, which in turn results in its ubiquination,

endocytosis and degradation through theproteasomal/lysosom-

al pathways (Dikic and Giordano, 2003). As Spry proteins

phosphorylated on Tyr 55 can also interact with c-Cbl, they are

in direct competition with activated EGFRs for binding to this

protein. As a result, the EGFR is not ubiquinated, internalized

or degraded in the presence of Spry, leading to prolonged cell-

surface exposure of the receptor and sustained signaling

activity. Therefore, in this context, Sprys function as positive

regulators of the EGF signaling resulting in an upregulation of

the MAP kinase ERK activity. A similar degradation of Spry2

by c-Cbl may also be true for FGFR signaling (Hall et al.,

2003).

However, most of the studies performed on FGF signaling

pathway show that Sprys associate with Grb2. For example,

mouse Spry2 and Xspry1 can interact with the SH2 domain of

Grb2 after FGFR-induced phosphorylation of Tyr55(Hanafusa

et al., 2002). As a result, the interaction of Grb2 with FRS2 is

blocked and FGF-induced signal transduction is repressed. Inaddition, Spry2 and 4 have been shown to bind directly to Raf

through a Raf-binding motif in their C-terminal domain and

suppress phosphorylation of Raf on Ser338 by the Raf kinase

PKCy (Sasaki et al., 2003).

Altogether, Sprys function through different pathways,

which determine the overall effect on RTK signaling. First,

binding of c-Cbl to tyrosine-phosphorylated Spry results in the

stabilization of EGFR and in sustained ERK activity. Second,

binding of Grb2 to tyrosine-phosphorylated Spry can interrupt

the FGFR/Ras/Raf/MAPK pathway upstream of Ras. Finally,

binding of the C-terminal domain of Spry by its Raf-binding

motif inhibits Raf phosphorylation by PLCg-activated PKCy.

However, the criteria to decide between Spry different

Fig. 2. FGF signaling regulates osteogenesis. During intramembranous bone development, bone formation is characterized by the proliferation of mesenchymal cells

followed by their commitment to become osteoprogenitor cells. They differentiate into preosteoblasts then into bone matrix-forming mature osteoblasts. FGFs are

required for each step of bone formation through FGFRs to control various genes involved in osteoblast commitment, differentiation and apoptosis. ALP: alkaline

phosphatase, BSP: bone sialoprotein, COL I: collagen type I, OC: osteocalcin, OP: osteospondin, ON: osteonectin (adapted from P. J.Marie, 2003).



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Fig. 3. Models for the attenuation of FGF signaling by Sprys. (I) Spry acts downstream of the FGFR and upstream of Ras by binding to the adaptor protein Grb2 at the le

the activation of Ras and the subsequent stimulation of the downstream elements of the pathway. (II) Spry also acts downstream of Ras by directly binding Raf. This is ach

located in its C-terminal part and this prevents both activation by Ras and phosphorylation of Ser338 by the PKC y. This explains how spry can inhibit the transduct


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functions are not yet known and will have to be defined in the

near future.

Other FGF synexpression group members

In addition to sprys, several other genes involved in the

regulation of the FGF pathway have been isolated through thecourse of large-scale in situ hybridization screens (Gawantka et

al., 1998; Thisse et al., 2001; Kudoh et al., 2001) designed to

identify genes showing expression pattern similar or over-

lapping with FGFs. Various genes were isolated such as

XFLRT3 (Bottcher et al., 2004), Sef (Furthauer et al., 2002;

Tsang et al., 2002), the MAP kinase phosphatase 3 (MKP3,

also called Pyst1, Eblaghie et al., 2003; Tsang et al., 2004),

PEA3 (for polyoma virus enhancer activator 3) and ERM

(Munchberg et al., 1999). These last two factors are the

downstream players of the FGF signaling pathway while the

others, also induced by FGF stimulation are involved in the

attenuation of the signal (for review, Tsang and Dawid, 2004).

XFLRT3

This gene encodes a transmembrane protein characterized

by a cluster of leucine-rich repeats and one FNIII domain

within the extracellular region. Its expression is induced after

activation of FGF signaling and downregulated after inhibition

of this pathway. XFLRT3 signaling results in phosphorylation

of the MAP kinase ERK and is blocked by the MAP kinase

phosphatase 1. In gain- and loss-of-function experiments

performed in Xenopus, FLRT3 was shown to phenocopy the

Fig. 4. FGF signaling and its regulation. The stimulation of FGFR by FGF ligands results in the activation of specific target genes. Among them are several feedback

inhibitors which are involved in the attenuation of FGF signaling. Spry acts at the level of Grb2 and/or at the level of Raf. Sef and XFLRT3 are both located at the

membrane and interact with FGFR. Sef functions as a negative regulator while XFLRT3 enhances the FGF activity resulting in the phosphorylation of the MAP

kinase ERK. Sef was shown also to affect the phosphorylation of the MAP kinase, either directly at the level of ERK or by preventing the phosphorylation of ERK byMEK. Finally, MKP3 negatively regulates the FGF signaling pathway by dephosphorylation of activated MAP kinase.



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FGF signaling (Bottcher et al., 2004). Surprisingly, in zebra-

fish, although FLRT3 is expressed in well known centers of

FGF signaling, neither gain nor loss of FGF signaling appears

to directly affect FLRT3 expression. Inactivating FLRT3

through injection of antisense morpholinos impairs conver-

gence-extension movements that direct axial elongation but

fails to mimick phenotypes observed after either loss or gainfunction of FGF activity. These observations suggest that, in

zebrafish, FLRT3 may be involved in directing cell movements

but does not appear to be related to the FGF pathway

(Furthauer M., Thisse, B. and Thisse, C., unpublished data).

Sef

The Sef protein is conserved across zebrafish, mouse and

human but not in invertebrates. Homology searches with Sef

revealed that it encodes a putative transmembrane protein, with

weak similarities to the Interleukin-17 receptor and a putative

tyrosine phosphorylation site into the intracellular region(Furthauer et al., 2002; Tsang et al., 2002). In zebrafish, loss-

or gain-of-function studies revealed Sef to function as an

antagonist of FGF signaling and to interfere with FGF signal

transduction by acting dowstream or at the level of MEK and

upstream (or at the level) of the MAP kinase (Furthauer et al.,

2002). Further studies performed using human Sef (hSef)

showed that it binds to activated forms of MEK, inhibits the

dissociation of the MEK ERK complex and blocks nuclear

translocation of activated ERK. Consequently, hSef inhibits

phosphorylation and activation of the nuclear ERK substrate

Elk-1, while it does not affect phosphorylation of the

cytoplasmic ERK substrate RSK2. Downregulation of endog-

enous hSef by hSef siRNA enhances the stimulus-induced

ERK nuclear translocation and the activity of Elk-1. These

results favor the hypothesis that hSef acts as a spatial regulator

for ERK signaling by targeting ERK to the cytoplasm (Toriiet

al., 2004). Other reports showed that the intracellular domain of

Sef is required for its inhibitory function and its interaction

with FGFR1 and FGFR2 (Tsang et al., 2002; Kovalenko et al.,

2003, Xiong et al., 2003). Ectopic expression of Sef blocks the

phosphorylation of FRS2. The conclusion of these studies was

that Sef acts at the level of FGFRs blocking the Ras and PI3K

pathways downstream. To complicate the further understanding

of Sef function, alternative spliced isoforms of Sef were found

in human (Preger et al., 2004). Sef-b which lacks the signalpeptide is localized in the cytoplasm and can suppress FGF

signaling downstream of MEK. Because FGFs deliver multiple

biological responses, it is possible that like Spry, Sef can inhibit

the FGF pathway at various points depending on the context, in

order to allow fine-tuning of signal regulation.

MAP kinase phosphatase 3 (MKP3)

Two distinct domains are characteristic of MKP3 proteins,

the N-terminal region, which contains a high-affinity ERK/

MAPK binding domain and the C-terminal domain, which

constitute a dual specificity phosphatase (Stewart et al., 1999;

Zhang et al., 2003; Zhao and Zhang, 2001). MKP3 negatively

regulates the MAP kinase cascade through dephosphorylation

of activated MAPK proteins. Upon binding of phosphorylated

MAPKs to the MAPK-binding domain in MKP3, a conforma-

tional activation of the C-terminal phosphatase domain is

achieved, leading to the inactivation of MAPKs (Camps et al.,

2000; Fjeld et al., 2000; Zhao and Zhang, 2001). In chicken,

ectopic expression of MKP3 results in the disruption of limboutgrowth, a phenotype characteristic of FGF signaling

blockage (Kawakami et al., 2003; Eblaghie et al., 2003). In

zebrafish, MKP3 limits the extent of FGF activity of the

gastrula embryo(Tsang et al., 2004).

Concluding remarks

A substantial amount of important work has been done to

establish the role of FGF signaling during development showing

that FGFs regulate cell fate and specification of germ layers.

FGF downstream elements have been characterized and it

becomes evident that a growing number of these factors arethemselves tightly regulated by FGFs. It is clear from various

studies that multiple feedback inhibitors exist and that they act at

different levels of the signal transduction cascades to attenuate

FGF signal(Fig.4). Regulation can occur at different levels of

the signal transduction pathways including the membrane down

to the level of phosphorylation of the MAP kinase. Some of the

feedback inhibitors are involved in more than one process (Sef,

Spry) and negatively regulate other RTK signaling pathways.

The next challenge will be to integrate the intricate

interactions between these different regulators within the

FGF pathway and between different RTK pathways. In the

future, it will be important to assess the pathways employed by

FGFs in different cells and define the determinants for

signaling specificity. This can include feedback regulation,

threshold-type responses and posttranslational modifications.

This will be complicated by the fact that these modulators

combine a wide range of activity. The complete understanding

of these mechanisms will have diverse implications in biology

as well as in therapeutic approaches.

Acknowledgment

We thank the ?FGF communityXfor help and apologize for

not having being able to cite as many papers as we would have

wished because of limited space.

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