Fibroblast Growth Factors (FGFs) are signaling molecules that are important in patterning and growth control during vertebrate limb development. Beads soaked in FGF-1, FGF-2 and FGF-4 are able to induce additional limbs when applied to the flank of young chick embryos (Cohn, M.J., Izpisua-Belmonte, J-C., Abud, H., Heath, J. K., Tickle, C. (1995) Cell 80, 739–746). However, biochemical and expression studies suggest that none of these FGFs is the endogenous signal that initiates limb development. During chick limb development, Fgf-8 transcripts are detected in the intermediate mesoderm and subsequently in the prelimb field ectoderm prior to the formation of the apical ectodermal ridge, structures required for limb initiation and outgrowth, respectively. Later on, Fgf-8 expression is restricted to the ridge cells and expression disappears when the ridge regresses. Application of FGF-8 protein to the flank induces the development of additional limbs. Moreover, we show that FGF-8 can replace the apical ectodermal ridge to maintain Shh expression and outgrowth and patterning of the developing chick limb. Furthermore, continuous and widespread misexpression of FGF-8 causes limb truncations and skeletal alterations with phocomelic or achondroplasia phenotype. Thus, FGF-8 appears to be a key signal involved in initiation, outgrowth and patterning of the developing vertebrate limb.

Reference

Abraham
J. A.
,
Mergia
A.
,
Whang
J. L.
,
Tumolo
A.
,
Friedman
J.
,
Hjerrild
K. A.
,
Gospodarowicz
D.
,
Fiddes
J. C.
(
1986
)
Nucleotide sequence of a bovine clone encoding the angiogenic protein, basic fibroblast growth factor.
Science
233
,
545
553
Amaya
E.
,
Musci
T. J.
,
Kirschner
M. W.
(
1991
)
Expression of a dominant negative mutant of the FGF receptor disrupts mesoderm formation in Xenopus embryos.
Cell
66
,
257
270
Bellus
G. A.
,
Hefferon
T. W.
,
Ortiz de Luna
R. I.
,
Hecht
J. T.
,
Horton
W. A.
,
Machado
M.
,
Kaitila
I.
,
McIntosh
I.
,
Francomano
C. A.
(
1995
)
Achondroplasia is defined by recurrent G380R mutations of FGFR3.
Am. J. Hum. Genet
56
,
368
373
Carrington
J. L.
,
Fallon
J. F.
(
1988
)
Initial limb budding is independent of apical ectodermal ridge activity; evidence from a limbless mutant.
Development
104
,
361
367
Chan
D. V.
,
Wynshaw-Boris
A.
,
Leder
P.
(
1995
)
Formin isoforms are differentially expressed in the mouse embryo and are required for normal expression of fgf-4 and shh in the limb bud.
Development
121
,
3151
3162
Chang
D. T.
,
Lopez
A.
,
vonKessler
D. P.
,
Chiang
C.
,
Simandl
B. K.
,
Zhao
R.
,
Seldin
M. F.
,
Fallon
J. F.
,
Beachy
P. A.
(
1994
)
Products genetic linkage and limb patterning activity of a murine hedgehog gene.
Development
120
,
3339
3353
Charite
J.
,
de Graaff
W.
,
Shen
S.
,
Deschamps
J.
(
1994
)
Ectopic expression of Hox b-8 causes duplication of the ZPA in the forelimb and homeotic transformation of axial structures.
Cell
78
,
589
601
Cho
K. W. Y.
,
De Robertis
E.
(
1990
)
Differential activation in Xenopus homeobox genes by mesoderm-inducing growth factors and retinoic acid.
Genes Dev
4
,
1910
1916
Cohn
M. J.
,
Izpisúa Belmonte
J. C.
,
Abud
H.
,
Heath
J. K.
,
Tickle
C.
(
1995
)
FGF-2 application can induce additional limbud, formation from the flank of chick embryos.
Cell
80
,
739
746
Crossley
P. H.
,
Martin
G. R.
(
1995
)
The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo.
Development
121
,
439
451
Dolle
P.
,
Dierich
A.
,
LeMeur
M.
,
Shimmang
T.
,
Schuhbaur
B.
,
Chambon
P.
,
Duboule
D.
(
1993
)
Disruption of the H oxd- 13 gene induces localized heterochrony leading to mice with neotenic limbs.
Cell
75
,
431
441
Dono
R.
,
Zeller
R.
(
1994
)
Cell-type-specific nuclear translocation of fibroblast growth factor-2 isoforms during chicken kidney and limb morphogenesis.
Dev. Biol
163
,
316
330
Duboule
D.
(
1994
)
How to make a limb?.
Science
266
,
575
576
Fallon
J.
,
Lopez
A.
,
Ros
M.
,
Savage
M.
,
Olwin
B.
,
Simandl
B.
(
1994
)
FGF-2, Apical ectodermal ridge growth signal for chick limb development.
Science
264
,
104
107
Geduspan
J. S.
,
Solursh
M.
(
1992
)
A growth promoting influence from the mesonephros during limb outgrowth.
Dev. Biol
151
,
242
250
Globus
M.
,
Vethamany-Globus
S.
(
1976
)
An in vitro analogue of early chick limb bud outgrowth.
Differentiation
6
,
91
96
Hamburger
V.
(
1938
)
Morphogenetic and axial self-differentiation of transplanted limb primordia of 2-day chick enmbryos.
J. Exp. Zool
77
,
379
400
Hamburger
V.
,
Hamilton
H.
(
1951
)
A series of normal stages in the development of the chick embryo.
J. Morph
88
,
49
92
Heikinheimo
M.
,
Lawshe
A.
,
Shackleford
G. M.
,
Wilson
D. B.
,
MacArthur
C. A.
(
1994
)
Fgf-8 expression in the post-gastrulation mouse suggests roles in the development of the face, limbs and central nervous system.
Mech. Dev
48
,
129
138
Hughes
S.
,
Greenhouse
J.
,
Petropoulos
J.
,
Sutrave
P.
(
1987
)
Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors.
J. Virology
61
,
3004
3012
Izpisúa Belmonte
J. C.
,
Brown
J. M.
,
Duboule
D.
,
Tickle
C.
(
1992
)
Expression of Hox-4 genes in the chick wing links pattern formation to the epithelial-mesenchymal interactions that mediate growth.
EMBO J
11
,
1451
1457
Izpisúa Belmonte
J. C.
,
Tickle
C.
,
Dolle
P.
,
Wolpert
L.
,
Duboule
D.
(
1991
)
Expression of the homeobox Hox-4 genes and the specification of position in chick wing development.
Nature
350
,
585
589
Jabs
E. W.
,
Li
X.
,
Scott
A. F.
,
Meyers
C.
,
Chen
W.
,
Eccles
M.
,
Mao
G.
,
Chamas
L. A.
,
Jackson
C. E.
,
Jaye
M.
(
1994
)
Jackson-Weiss and Crouzon syndromes are allelic with mutations in fibroblast growth factor receptor 2.
Nature Genetics
8
,
275
279
Jackson
A.
,
Friedman
S.
,
Zhan
X.
,
Engleka
K. A.
,
Forough
A.
,
Maciag
T.
(
1992
)
Heat shock induces the release of fibroblast growth factor 1 from NIH3T3 cells.
Proc. Natl. Acad. Sci. USA
89
,
10691
10695
Johnson
D. E.
,
Lee
P. L.
,
Lu
J.
,
Williams
L. T.
(
1990
)
Diverse forms of a receptor for acidic and basic fibroblast growth factor.
Mol. Cell. Biol
10
,
4728
4736
Koyama
E.
,
Noji
S.
,
Nohno
T.
,
Myokai
F.
,
Ono
K.
,
Nishijima
K.
,
Kuroiwa
A.
,
Ide
H.
,
Taniguchi
S.
,
Saito
T.
(
1993
)
Cooperative activation of HoxD homeobox genes by factors from the polarizing region and the apical ridge in chick limb morphogenesis.
Dev. Growth. Diff
35
,
189
198
Lajeunie
E.
,
Ma
H. W.
,
Bonaventure
J.
,
Munnich
A.
,
Le Merrer
M.
(
1995
)
FGFR-2 mutations in Pfeiffer syndrome.
Nature Genet
9
,
108
–.
Laufer
E.
,
Nelson
C. E.
,
Johnson
R. L.
,
Morgan
B. A.
,
Tabin
C.
(
1994
)
Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud.
Cell
79
,
993
1003
Lopez-Martinez
A.
,
Chang
D. T.
,
Chiang
C.
,
Porter
J. A.
,
Ros
M. A.
,
Simandl
B. K.
,
Beachy
P. A.
,
Fallon
J. F.
(
1995
)
Limb-patterning activity and restricted posterior localization of the amino-terminal product of Sonic hedgehog cleavage.
Current Biology
5
,
791
796
Mahmood
R.
,
Bresnick
J.
,
Hornbruch
A.
,
Mahony
C.
,
Morton
N.
,
Colquhoun
K.
,
Martin
P.
,
Lumsden
A.
,
Dickson
C.
,
Mason
I.
(
1995
)
A role for FGF-8 in the initiation and maintenance of vertebrate limb bud outgrowth.
Curr. Biol
5
,
797
806
Mansukhani
A.
,
Moscatelli
D.
,
Talarico
D.
,
Levytska
V.
,
Basilico
C.
(
1990
)
A murine fibroblast growth factor (FGF) receptor expressed in CHO cells is activated by basic FGF and Kaposi FGF.
Proc. Natl. Acad. Sci. USA
87
,
4378
4382
Mills
C. L.
,
Bellairs
R.
(
1989
)
Mitosis and cell death in the tail of the chick embryo.
Anatomy and Embryology
180
,
301
308
Mima
T.
,
Ohuchi
H.
,
Noji
S.
,
Mikawa
T.
(
1995
)
FGF can induce outgrowth of somatic mesoderm both inside and outside of limb-forming regions.
Dev. Biol
167
,
617
20
Miyamoto
M.
,
Naruo
K. I.
,
Sero
C.
,
Matsumoto
S.
,
Kondo
T.
,
Kurokawa
T.
(
1993
)
Molecular cloning of a novel cytokine cDNA encoding the ninth member of the fibroblast growth factor family, which has a unique secretion property.
Mol. Cell. Biol
13
,
4251
4259
Morgan
B. A.
,
Izpisúa-Belmonte
J.-C.
,
Duboule
D.
,
Tabin
C. J.
(
1992
)
Targeted misexpression of Hox-4. 6 in the avian limb bud causes apparent homeotic transformations.
Nature
358
,
236
239
Niswander
L.
,
Martin
G. R.
(
1992
)
FGF-4 expression during gastrulation, myogenesis, limb and tooth development in the mouse.
Development
114
,
755
768
Niswander
L.
,
Martin
G. R.
(
1993
)
FGF-4 and BMP-2 have opposite effects on limb outgrowth.
Nature
361
,
68
76
Niswander
L.
,
Jeffery
S.
,
Martin
G.
,
Tickle
C.
(
1994
)
Signaling in vertebrate limb development: a positive feedback loop between sonic hedgehog and FGF 4.
Nature
371
,
609
612
Niswander
L.
,
Tickle
C.
,
Vogel
A.
,
Martin
G.
(
1994
)
Function of FGF-4 in limb development.
Mol. Reprod. Dev
39
,
83
89
Niswander
L.
,
Tickle
C.
,
Vogel
A.
,
Booth
I.
,
Martin
G. R.
(
1993
)
FGF-4 replaces the apical ectodermal ridge and directs outgrowth and patterning of the limb.
Cell
75
,
579
587
Ohuchi
H.
,
Nakagawa
T.
,
Yamaguchi
M.
,
Ohata
T.
,
Yoshioka
H.
,
Kuwana
T.
,
Mima
T.
,
Miwaka
T.
,
Nohno
T.
,
Noji
S.
(
1995
)
An additional limb can be induced from the flank of the chick embryo by FGF4.
Biochem. and Biophys. Res. Comm
209
,
809
816
Ohuchi
H.
,
Yoshioka
H.
,
Tanaka
A.
,
Kawakami
Y.
,
Nohno
T.
,
Noji
S.
(
1994
)
Involvement of androgen-induced growth factor (FGF-8) gene in mouse embryogenesis and morphogenesis.
Biochem. Biophys. Res. Commun
204
,
882
888
Orr-Urtreger
A.
,
Bedford
M. T.
,
Burakova
T.
,
Arman
E.
,
Zimmer
Y.
,
Yayon
A.
,
Givol
D.
,
Lonai
P.
(
1993
)
Developmental localization ofthe splicing alternatives of fibroblast growth factor receptor-2 (FGFR-2).
Dev Biol
158
,
475
486
Orr-Urtreger
A.
,
Givol
D.
,
Yayon
A.
,
Yarden
Y.
,
Lonai
P.
(
1991
)
Developmental expression of two murine fibroblast growth factor receptors, flg and bek.
Development
113
,
1419
1434
Peters
K. G.
,
Werner
S.
,
Chen
G.
,
Williams
L. T.
(
1992
)
Two FGF receptor genes are differentially expressed in epithelial and mesenchymal tissues during limb formation and organogenesis in the mouse.
Development
114
,
233
243
Peters
K.
,
Ornitz
D.
,
Werner
S.
,
Williams
L.
(
1993
)
Unique expression pattern of the FGF-receptor 3 gene during mouse organogenesis.
Dev. Biol
155
,
423
430
Reardon
W.
,
Winter
R. M.
,
Rutland
P.
,
Pulleyn
L. J.
,
Jones
B. M.
,
Malcom
S.
(
1994
)
Mutations in the fibroblast growth factor receptor 2 cause Crouzon syndrome.
Nature Genet
8
,
98
103
Reiter
R. S.
,
Solursh
M.
(
1982
)
Mitogenic property of the apical ectodermal ridge.
Dev. Biol
93
,
28
35
Riddle
R. D.
,
Ensini
M.
,
Nelson
C.
,
Tsuchida
T.
,
Jessel
T. M.
,
Tabin
C.
(
1995
)
Induction of the LIM homeobox gene Lmx1 by WNT7a establishes dorsoventral pattern in the vertebrate limb.
Cell
83
,
631
640
Riddle
R. D.
,
Johnson
R. L.
,
Laufer
E.
,
Tabin
C.
(
1993
)
Sonic hedgehog mediates the polarizing activity of the ZPA.
Cell
75
,
1401
1416
Riley
B. B.
,
Savage
M. P.
,
Simandl
B. K.
,
Olwin
B. B.
,
Fallon
J. F.
(
1993
)
Retroviral expression of FGF-2 (bFGF) affects patterning in the chick limb bud.
Development
118
,
95
104
Rousseau
F.
,
Bonaventure
J.
,
Legeal-Mallet
L.
,
Pelet
A.
,
Rozet
J.-M.
,
Maroteaux
P.
,
Le Merrer
M.
,
Munnich
A.
(
1994
)
Mutations in the gene encoding fibroblast growth factor receptor-3 in achondroplasia.
Nature
371
,
252
254
Rubin
L.
,
Saunders
J. W. J.
(
1972
)
Ectodermal-mesodermal interactions in the growth of limb buds in the chick embryo: constancy and temporal limits of the ectodermal induction.
Dev. Biol
28
,
94
112
Ruiz i Altaba
A.
,
Melton
D. A.
(
1989
)
Interaction between growth factors and homeobox genes in the establishment of anterior-posterior polarity in frog embryos.
Nature
341
,
33
38
Rutland
P.
,
Pulleyn
L. J.
,
Reardon
W.
,
Baraisler
M.
,
Hayward
R.
,
Jones
B.
,
Malcom
S.
,
Winter
R. M.
,
Oldridge
M.
,
Slaney
S. F.
(
1995
)
: Identical mutations in the FGFR-2 gene cause both Pfeiffer and Crouzon syndrome phenotypes.
Nature Genet
9
,
173
176
Saunders
J. W.
Jr
(
1948
)
The proximo-distal sequence of the origin of the parts of the chick wing and the role of the ectoderm.
J. Exp. Zool
108
,
363
404
Saunders
J. W.
Jr
(
1972
)
Developmental control of three-dimensional polarity in the avian limb.
Ann. NY Acad. Sci
193
,
29
42
Saunders
J. W.
Jr.
,
Reuss
C.
(
1974
)
Inductive and axial properties of prospective wing-bud mesoderm in the chick embryo.
Dev. Biol
38
,
41
50
Savage
M. P.
,
Hart
C. E.
,
Riley
B. B.
,
Sasse
J.
,
Olwin
B. B.
,
Fallon
J. F.
(
1993
)
Distribution of FGF-2 suggests it has a role in chick limb bud growth.
Dev. Dyn
198
,
159
170
Searls
R. L.
,
Janners
M. Y.
(
1971
)
The initiation of limb bud outgrowth in the embryonic chick.
Dev. Biol
24
,
198
213
Searls
R. L.
,
Zwilling
E.
(
1964
)
Regeneration of the apical ectodermal ridge of the chick limb bud.
Dev. Biol
9
,
35
55
Selleck
M. A. J.
,
Stern
C. D.
(
1992
)
Commitment of mesoderm cells in Hensen's node of the chick embryo to notochord and somite.
Development
114
,
403
415
Shiang
R.
,
Thompson
L. M.
,
Zhu
Y.-Z.
,
Church
D. M.
,
Fielder
T. J.
,
Bocian
M.
,
Winokur
S. T.
,
Wasmuth
J. J.
(
1994
)
Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia.
Cell
78
,
335
342
Slack
J. M. W.
(
1995
)
Growth factor lends a hand.
Nature
374
,
217
218
Smith
D. W.
,
Jones
K. L.
(
1982
)
Recognizable patterns of human malformation: Genetic embryologic and clinical aspects.
(ed.
Markowitz
M.
)
Series Title Major Problems In Clinical Pediatrics.
Stephens
T. D.
,
Spall
R.
,
Baker
W. C.
,
Hiatt
S. R.
,
Pugmire
D. E.
,
Shaker
M. R.
,
Willis
H. J.
,
Winger
K. P.
(
1991
).
Axial and paraxial influences on limb morphogenesis.
J. Morphol
208
,
367
379
Stoilov
I.
,
Kilpatrick
M. W.
,
Tsipouras
P.
(
1995
)
A common FGFR3 gene mutation is present in achondroplasia but not in hypochondroplasia.
Am. J. Med. Genetics
55
,
127
133
Summerbell
D.
(
1974
)
A quantitative analysis of the effect of excision of the AER from the chick limb bud.
J. Embryol. Exp. Morph
32
,
651
660
Superti-Furga
A.
,
Eich
G.
,
Bucher
H. U.
,
Wisser
J.
,
Giedon
A.
,
Gitzelmann
R.
,
Steinmann
B.
(
1995
)
A glycine 375-to-cysteine substitution in the transmembrane domain of the fibroblast growth factor receptor-3 in a newborn with achondroplasia.
Eur J. Pediatr
154
,
215
219
Suzuki
H. R.
,
Sakamoto
H.
,
Yoshida
T.
,
Sugimura
T.
,
Terada
M.
,
Solursh
M.
(
1992
)
Localization of Hst1 transcripts to the apical ectodermal ridge in the mouse embryo.
Dev. Biol
150
,
219
222
Tabin
C.
(
1995
)
The initiation of the limb bud: Growth factors, Hox genes, and retinoids.
Cell
80
,
671
674
Tanaka
A.
,
Miyamoto
K.
,
Minamino
N.
,
Takeda
M.
,
Sato
B.
,
Matsuo
H.
,
Matsumoto
K.
(
1992
)
Cloning and characterization of an androgen-induced growth factor essential for the andorgen-dependent growth of mouse mammary carcinoma cells.
Proc. Natl. Acad. Sci. USA
89
,
8928
8923
Tavormina
P. L.
,
Shiang
R.
,
Thompson
L. M.
,
Zhu
Y. Z.
,
Wilkin
D. J.
,
Lachman
R. S.
,
Wilcox
W. R.
,
Rimoin
D. L.
,
Cohn
D. H.
,
Wasmuth
J. J.
(
1995
)
Thanatohoric dysplasia (types I and II) caused by distinct mutations in fibroblast growth factor receptor 3.
Nature Genet
9
,
231
328
Tickle
C.
,
Eichele
G.
(
1994
)
Vertebrate limb development.
Ann. Rev. Cell Biol
10
,
121
52
Vogel
A.
,
Tickle
C.
(
1993
)
FGF-4 maintains polarizing activity of posterior limb bud cells in vivo and in vitro.
Development
119
,
199
206
Vogel
A.
,
Roberts-Clarke
D.
,
Niswander
L.
(
1995
)
Effect of FGF on gene expression in chick limb bud cells in vivo and in vitro.
Dev. Biol
171
,
507
520
Vogel
A.
,
Rodriguez
C.
,
Warnken
W.
,
Izpisua-Belmonte
J. C.
(
1995
)
Chick Lmx-1 specifies dorsal cells fate during vertebrate limb development.
Nature
378
,
716
720
Werner
S.
,
Duan
D.-s. R.
,
De Vries
C.
,
Peters
K. P.
,
Johnson
D.
,
Williams
L. T.
(
1992
)
Differential splicing in the extracellular region of fibroblast growth factor receptor 1 generates receptor variants with different ligand-binding specificities.
Mol. Cell. Biol
12
,
82
88
Wilkie
A. O. M.
,
Morriss-Kay
G. M.
,
Jones
E. Y.
,
Heath
J. K.
(
1995
)
Functions of fibroblast growth factors and their receptors.
Current Biology
5
,
500
507
Wilkie
A. O. M.
,
Slaney
S. F.
,
Oldridge
M.
,
Poole
M. D.
,
Ashworth
G. J.
,
Hockley
A. D.
,
Hayward
R. D.
,
David
D. J.
,
Pulleyn
L.
,
Rutland
P.
(
1995
)
Apert syndrome results from localized mutations of FGFR-2 and is allelic to Crouzon syndrome.
Nature Genet
9
,
165
172
Yang
Y.
,
Niswander
L.
(
1995
)
Interaction between the signaling molecule WNT7a and SHH during vertebrate limb development: dorsal signals regulated anteroposterior patterning.
Cell
80
,
939
47
Zwilling
E.
(
1961
)
Limb morphogenesis.
Adv. Morph
1
,
301
330
Zwilling
E.
(
1972
)
Limb morphogenesis.
Dev. Biol.
28
,
12
17
This content is only available via PDF.