Fibroblast Growth Factor Signaling during Early Vertebrate Development
Ralph T.Bo
¨ttcher and Christof Niehrs Division of Molecular Embryology,Deutsches Krebsforschungszentrum,D-69120Heidelberg,Germany
Fibroblast growth factors (FGFs)have been implicated in di-verse cellular processes including apoptosis,cell survival,
chemotaxis,cell adhesion,migration,differentiation,and pro-liferation.This review presents our current understanding on the roles of FGF signaling,the pathways employed,and its
regulation.We focus on FGF signaling during early embryonic processes in vertebrates,such as induction and patterning of the three germ layers as well as its function in the control of morphogenetic movements.(Endocrine Reviews 26:63–77,2005)
甲苯二异氰酸酯用途I.Introduction
II.FGFs and FGF Receptors (FGFRs)III.FGFR Signal Transduction
A.Ras/MAPK pathway
B.PLC ␥/Ca 2ϩpathway
C.PI3kinase/Akt pathway IV.The Biological Activities of FGFs
A.FGF signaling pathway in mesoderm formation and
gastrulation movements
B.Chemotactic action of FGFs in morphogenetic movements
C.FGF signaling in neural induction and AP patterning
ddcD.FGF signaling in endoderm formation V.Regulation of FGF Signaling
A.Regulation by HSPGs
B.Transmembrane regulators
C.Intracellular regulators VI.Concluding Remarks
I.Introduction
F
IBROBLAST GROWTH FACTORS (FGFs)constitute a large family of polypeptide growth factors found in a variety of multicellular organisms,including invertebrates.The first member of this family,FGF2,was identified by its ability to stimulate proliferation of mouse 3T3fibroblasts (1);however,the function of FGFs is not restricted to cell growth.Instead,FGFs are involved in diverse cellular processes in-cluding chemotaxis,cell migration,differentiation,cell sur-vival,and apoptosis.The common feature of FGFs is that they are structurally related and generally signal through receptor tyrosine kinases.FGFs play an important role in
embryonic development in invertebrates and vertebrates.This review focuses on the role of FGF signaling during early vertebrate development.Excellent reviews dealing with the role of FGFs in Drosophila (2–4),Caenorhabditis elegans (5,6),vertebrate limb development (7–9),central nervous system development (10,11),skeleton formation (12),and cancer (13–15)are available.
II.FGFs and FGF Receptors (FGFRs)
The human FGF protein family consists of 22members that share a high affinity for heparin as well as a high-sequence homology within a central core domain of 120amino acids (16),which interacts with the FGFR (17–19)(Fig.1A).FGFs can be classified into subgroups according to structure,bio-chemical properties,and expression (for reviews see Refs.16and 20).For example,members of the FGF8subfamily (FGF8,FGF17,FGF18)share 70–80%amino acid sequence identity and similar receptor-binding properties and have overlap-ping expression patterns.FGFs appear to be diffusible (21),and they are able to act in a dose-dependent fashion,e.g .to induce different marker genes at different concentrations (22,23).This raises the question of whether FGFs act physiolog-ically as morphogens.Indeed,in neural progenitors,graded FGF signaling appears to be translated into a distinct motor neuron Hox pattern (24).
FGFs induce their biological responses by binding to and activating FGFRs,a subfamily of cell surface receptor ty-rosine kinases (RTKs).The vertebrate Fgfr gene family con-sists of four highly related genes,Fgfr1–4(25).These genes code for single spanning transmembrane proteins with an extracellular ligand-binding region and an intracellular do-main harboring tyrosine kinase activity.The extracellular region is composed of Ig-like domains that are required for FGF binding.The Ig-like domains also regulate binding af-finity and ligand specificity (Fig.1B).Located between Ig-like domain
s I and II is a stretch of acidic amino acids (acidic box domain)followed by a heparin-binding region and a cell adhesion homology domain.These domains are required for the interaction of the receptor with components of the ex-First Published Online October 26,2004
Abbreviations:AP,Anterior-posterior;BMP,bone morphogenetic protein;CAM,cell adhesion molecule;EMT,epithelial to mesenchymal transition;FGF,fibroblast growth factor;FGFR,FGF receptor;HS,hepa-ran sulfate;HSPG,heparan sulfate proteoglycan;MO,morpholino oli-gonucleotide;PI3,phosphatidylinositol-3;PLC,phospholipase C;PTP,protein tyrosine phosphatase;RTK,receptor tyrosine kinase;SOS,Son of sevenless.
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tracellular matrix,in particular heparan sulfate (HS)proteo-glycans (HSPGs),and cell adhesion molecules (CAMs).The intracellular part of the receptor includes the juxtamembrane domain,the split tyrosine kinase domain,and a short carboxy-terminal tail.In addition to the enzymatic activity,the intracellular domain harbors protein binding and phos-phorylation sites (including protein kinase C and FRS2sites)as well as several autophosphorylation sites that interact with intracellular substrates.
Alternative splicing of Fgfr transcripts generates a diver-sity of FGFR isoforms (25,26).In vitro assays show that different isoforms have distinct FGF-binding specificities (27,28).The tissue-specific alternative splicing encompassing the carboxy-terminal half of Ig domain III strongly affects ligand-receptor binding specificity (29–31).In vivo ,FGFR-FGF com-plex assembly and signaling is further regulated by the spa-tial and temporal expression of endogenous HSPGs (32,33).HSPGs are required to activate effectively the FGFR and determine the interaction between specific FGF-FGFR pairs (see Section V.A.).
III.FGFR Signal Transduction
FGFRs,like other receptor tyrosine kinases,transmit ex-tracellular signals after ligand binding to vario
us cytoplas-mic signal transduction pathways through tyrosine phos-phorylation.Binding of FGFs causes receptor dimerization
and triggers tyrosine kinase activation leading to autophos-phorylation of the intracellular domain (34).Tyrosine auto-phosphorylation controls the protein tyrosine kinase activity of the receptor but also serves as a mechanism for assembly and recruitment of signaling complexes (35).These phos-phorylated tyrosines function as binding sites for Src ho-mology 2and phosphotyrosine binding domains of signaling proteins,resulting in their phosphorylation and activation (36,37).A subset of Src homology 2-containing proteins such as Src-kinase and phospholipase C ␥(PLC ␥)possesses in-trinsic catalytic activities,whereas others are adapter pro-teins.FGF signal transduction,as analyzed during early em-bryonic development,can proceed via three main pathways described below (Fig.2).
A.Ras/MAPK pathway
The most common pathway employed by FGFs is the MAPK pathway.This involves the lipid-anchored docking protein FRS2(also called SNT1)that constitutively binds FGFR1even without receptor activation (38–40).Several groups have demonstrated the importance of FRS2in FGFR1-mediated signal transduction during embryonic de-velopment (41–43).After activation of the FGFR,ty
rosine-phosphorylated FRS2functions as a site for coordinated as-sembly of a multiprotein complex activating and controlling the Ras-MAPK signaling cascade and the
phosphatidylino-
F I
G .1.Domain structure of generic FGF and FGFR proteins.A,Structure of a generic FGF protein containing a signal sequence and the
赫兹伯格
conserved core region that contains receptor-and HSPG-binding sites.B,The main structural features of FGFRs including Ig domains,acidic box,heparin-binding domain,CAM-homology domain (CHD),transmembrane domain,and a split tyrosine kinase domain are illustrated with respective functions.CAM,Cell adhesion molecule;ECM,extracellular matrix;PKC,protein kinase C.
64Endocrine Reviews,February 2005,26(1):63–77Bo ¨ttcher and Niehrs •FGF Signaling in Vertebrate Development
sitol 3(PI3)-kinase/Akt pathway.The FRS2tyrosine phos-phorylation sites are recognized and bound by the adapter protein Grb2and the protein tyrosine phosphatase (PTP)Shp2(39,44).Grb2forms a complex with the guanine nu-cleotide exchange factor Son of sevenless (SOS)via its SH3domain.Translocation of this complex to the plasma mem-brane by binding to phosphorylated F
RS2allows SOS to activate Ras by GTP exchange due to its close proximity to membrane-bound Ras.Once in the active GTP-bound state,
Ras interacts with several effector proteins,including Raf leading to the activation of the MAPK signaling cascade.This cascade leads to phosphorylation of target transcription fac-
tors,such as c-myc ,AP1,and members of the Ets family of transcription factors (reviewed in Ref.45).
B.PLC ␥/Ca 2ϩpathway
The PLC ␥/Ca 2ϩpathway involves binding of PLC ␥to phosphorylated tyrosine 766of FGFR1(46,47).Upon acti-vation,PLC ␥hydrolyzes phosphatidylinositol-4,5-diphos-phate to form two second messengers,inositol-1,4,5-triphos-phate and diacylglycerol.Diacylglycerol is an activator of protein kinase C,whereas inositol-1,4,5-triphosphate stim-ulates the release of intracellular Ca 2ϩ.This cascade has been
F I
G .2.Intracellular signaling pathways activated through FGFRs.For simplicity,only those proteins are indicated that are discussed in the main text.Formation of a ternary FGF-heparin-FGFR complex leads to receptor autophosphorylation and activation of intracellular signaling cascades,including the Ras/MAPK pathway (shown in blue ),PI3kinase/Akt pathway (shown in green ),and the PLC ␥/Ca 2ϩ
pathway (shown in yellow ).Proteins connected with two pathways are striped .Members of the FGF synexpression group are illustrated in red .The Ras/MAPK cascade is activated by binding of Grb2to phosphorylated FRS2.The subsequent formation of a Grb2/SOS complex leads to the activation of Ras.Three routes by which FGFRs can activate PI3kinase/Akt pathway are indicated.First,Gab1can bind to FRS2indirectly via Grb2,resulting in tyrosine phosphorylation and activation of the PI3-kinase/Akt pathway via p85(41,201).Second,the PI3kinase-regulatory subunit p85can bind to a phosphorylated tyrosine residue of the FGFR,as shown in Xenopus cell extracts (202).Alternatively,activated Ras can induce membrane localization and activation of the p110catalytic subunit of PI3kinase (203,204).AA,Arachidonic acid;DAG,diacylglycerol;IP 3,inositol-1,4,5-triphosphate.
Bo ¨ttcher and Niehrs •FGF Signaling in Vertebrate Development Endocrine Reviews,February 2005,26(1):63–7765
implicated in the FGF2-stimulated neurite outgrowth (48,49)
and in the caudalization of neural tissue by FGFR4in Xenopus (50).
C.PI3kinase/Akt pathway
The PI3kinase/Akt pathway can be activated by three mechanisms after FGFR activation (Fig.2),and the phos-pholipids thereby generated regulate directly or indirectly the activity of target proteins such as Akt/PKB.
Among other processes,the PI3kinase signaling branch is involved during Xenopus mesoderm induction acting in par-allel to the Ras/MAPK pathway.Overexpression of a domi-nant negative form of the PI3kinase-regulatory subunit p85interferes with Xenopus mesoderm formation.Conversely,coexpression of activated forms of MAPK and PI3kinase leads to synergistic mesoderm induction (51).
IV.The Biological Activities of FGFs
Early developmental processes during gastrulation stages of Xenopus ,zebrafish,chicken,and mouse include mesoderm formation and gastrulation movements,neural induction and AP patterning,and endoderm formation (Fig.3).FGF signaling plays important roles in early vertebrate develop-ment during these processes as discussed below and sum-marized in Table 1.Genes that function in FGF signaling and for which loss-of-function analysis revealed an early embry-onic phenotype are summarized in Table 2.
F I
fgf
G .3.Early developmental events during Xenopus ,zebrafish,chick,and mouse gastrulation.A,In Xenopus ,the three germ layers form along the animal-vegetal axis:the pigmented animal pole becomes ectoderm,and the yolk-rich vegetal pole becomes endoderm.Mesoderm is induced in an equatorial region (marginal zone)between the two other layers by signals emitted from the underlying prospective endoderm (black arrows ).The dorsal mesoderm,the Spemann organizer,emits neural inducing signals (blue arrow ).In the anamniotes,Xenopus and zebrafish (A and B),gastrulation is triggered by the involution of prospective mesendodermal cells starting at the organizer region of the embryos (red arrows ).Convergence of cells toward the embryonic midline and anterior-posterior elongation of the body axis is achieved by cellular rear-rangements (convergent extension).B,In zebrafish,the yolk syncitial layer (YSL)emits meso-and endoderm-inducing signals to the blastoderm (black arrows ).Close to the margin,mesodermal and endodermal progenitors are interspersed,whereas further away from the margin only mesodermal cells are present.The zebrafish organizer,the shield,emits neural inducing signals acting within the plane of the epiblast (blue arrow ).C,The chick embryo is a bilayered structure composed of the epiblast and the extraembryonic hypoblast.[Modified with permission from Elsevier from A.Locascio and M.A.Nieto:Curr Opin Genet D
ev 11:464–469,2001(205)].Gastrulation starts with the formation of the primitive streak with Hensen’s node (the avian organizer,Hn)forming at its tip.During gastrulation,epiblast cells undergo epithelial to mesenchymal transition and ingress as individual cells through the primitive streak (red arrows ).In the space between the epiblast and the hypoblast the migrating cells form axial and lateral mesoderm as well as definitive endoderm.Ectodermal cells become specified as either neural or epidermal cells before the onset of gastrulation.D,The mouse gastrula embryo has a cylindrical shape and consists of an outer and inner epidermal layer (for simplicity,only the inner layer is shown)[Adapted with permission from Elsevier from R.S.Beddington and J.C.Smith:Curr Opin Genet Dev 3:655–661,1993,(206).]As gastrulation starts,epiblast cells at the posterior side undergo an epithelial-mesenchymal transformation to generate mesoderm precursors and form the primitive streak.The streak elongates during gastrulation while mesodermal and endodermal progenitors start migrating through the primitive streak (red arrows ).
66Endocrine Reviews,February 2005,26(1):63–77Bo ¨ttcher and Niehrs •FGF Signaling in Vertebrate Development
A.FGF signaling pathway in mesoderm formation and gastrulation movements
Induction and patterning of mesoderm is one of the ear-liest events during vertebrate body axis formation.Although TGF-signaling plays a pivotal role in mesoderm induction and patterning and has received most attention in this con-text recently,FGF signaling is also essential.Indeed,the first identified mesoderm inducer was basic FGF(52),and this represents an evolutionarily conserved role,because FGF signaling is required for mesoderm induction in the primi-tive chordate Ciona(53).In Xenopus,mouse,and zebrafish, disruption of FGF signaling by Fgfr knock out or by over-expression of a dominant negative FGFR1strongly affects body axis formation(54–57).In these embryos,phenotypic changes are observed mostly in posterior regions,such as defects of trunk and tail structures.Despite some organism-specific differences,two functions for FGFs are conserved. First,FGFs control the specification and maintenance of me-soderm by regulation of T box transcription factors in Xe-nopus(58,59),mouse(60,61),and zebrafish(57,62,63). Second,they have a direct and/or indirect role in morpho-genetic movements during gastrulation.涓滴理论
The effect of FGFs on mesoderm formation was first dem-onstrated and most extensively studied in Xenopus(64).Since the first studies that discovered the mesoderm-inducing abil-ity of FGFs(52,65,66),many components of the FGF sig-naling pathway were shown in Xenopus to be required for mesoderm development.These components include FGFRs (54,67),HSPGs(68),adaptor
proteins FRS2/SNT-1,Nck,and Grb2(42,43,69),Src-kinase Laloo(70),PTPs(71,72),Ras,Raf, MAPK kinase,ERK(73–76),and the transcription factors AP1 and Ets2(77,78).Their inhibition blocks mesoderm forma-tion and induces posterior and gastrulation defects.Con-versely,in gain-of-function experiments they induce meso-dermal markers and phenocopy FGF overexpression.The role of FGF signaling during mesoderm formation is that of a competence factor.It is required for cells to respond to TGF--like mesoderm inducers,because activin-mediated mesoderm induction in Xenopus animal caps,for example,is blocked by a dominant negative FGFR1(79–81).Similarly, the trunk-inducing activity of dorsal mesoderm,which de-pends on Nodal signaling,is inhibited by a dominant neg-ative FGFR1(82).The ability of FGFs to regulate Nodal signaling may be due to phosphorylation of Smad2by MAPKs(83).
FGF signaling needs to continue during gastrulation for mesoderm maintenance as shown by transgenic Xenopus em-bryos overexpressing a dominant negative FGFR1(84).One of the FGF mesodermal targets is the T box transcription factor brachyury(Xbra),required for posterior mesoderm and axis formations in mouse,zebrafish,and Xenopus(58,85–87). Xbra and eFGF form an autoregulatory loop in which Xbra induces eFGF and vice versa(88,89).eFGF is also required for XmyoD expression in the myogenic cell lineage of Xenopus. eFGF is a candidate factor mediating th
e community effect, whereby cells differentiate into muscle once a critical cell mass is reached,due to the accumulation of diffusible factors (90,91).
T ABLE1.Processes involving FGF signaling in early vertebrate embryos
Source of FGFs Role Organism Ref.
I.Gastralation movements
Primitive streak(FGF8)and Hensen’s node(FGF4)Chemotactic signals coordinate
cell movements during
gastrulation
Chick114
Primitive streak EMT and migration of cells Mouse55,56,60,181
Mesodermal ring Convergent extension
movements
Xenopus89,92,95,97
II.Mesoderm formation
Epiblast and primitive streak Mesoderm formation and
patterning
Mouse60,61,108 Animal hemisphere and marginal zone Mesoderm formation and
patterning:competence factor
Xenopus81;reviewed in Ref.207 Marginal mesoderm DV patterning Zebrafish105
Trunk and tail development57,62
Competence factor63,98 III.AP patterning
Hensen’s node Maintenance of presumptive
neural progenitors to promote
development of posterior
nervous system
Chick151
tailbud?Hox gene regulation147
tailbud?AP patterning of mesoderm and
Hox gene regulation
Mouse146
Dorsolateral mesoderm(eFGF)AP patterning of mesoderm and
neuroectoderm
Xenopus50,125,126,131,136,137,149 Marginal mesoderm Posteriorization of neural tissue Zebrafish142
IV.Neural induction
Epiblast(FGF3),hypoblast(FGF8)Neural induction Chick118,119,208
Animal hemisphere Neural induction Xenopus23,125,126,132
V.Endoderm formation
Inner cell mass(FGF4)Endoderm formation Mouse152,154,155,157
Vegetal hemisphere Endoderm patterning Xenopus160–163
Bo¨ttcher and Niehrs•FGF Signaling in Vertebrate Development Endocrine Reviews,February2005,26(1):63–7767
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