In chick embryos homozygous for the limbless mutation, limb bud outgrowth is initiated, but a morphologically distinct apical ridge does not develop and limbs do not form. Here we report the results of an analysis of gene expression in limbless mutant limb buds. Fgf4, Fgf8, Bmp2 and Msx2, genes that are expressed in the apical ridge of normal limb buds, are not expressed in the mutant limb bud ectoderm, providing molecular support for the hypothesis that limb development fails in the limbless embryo because of the inability of the ectoderm to form a functional ridge. Moreover, Fgf8 expression is not detected in the ectoderm of the prospective limb territory or the early limb bud of limbless embryos. Since the early stages of limb bud outgrowth occur normally in the mutant embryos, this indicates that FGF8 is not required to promote initial limb bud outgrowth. In the absence of FGF8, Shh is also not expressed in the mutant limb buds, although its expression can be induced by application of FGF8-soaked beads. These observations support the hypothesis that Fgf8 is required for the induction of Shh expression during normal limb development. Bmp2 expression was also not detected in mutant limb mesoderm, consistent with the hypothesis that SHH induces its expression. In contrast, SHH is not required for the induction of Hoxd11 or Hoxd13 expression, since expression of both these genes was detected in the mutant limb buds. Thus, some aspects of mesoderm A-P patterning can occur in the absence of SHH and factors normally expressed in the apical ridge. Intriguingly, mutant limbs rescued by local application of FGF displayed a dorsalized feather pattern. Furthermore, the expression of Wnt7a, Lmx1 and En1, genes involved in limb D-V patterning, was found to be abnormal in mutant limb buds. These data suggest that D-V patterning and apical ridge formation are linked, since they show that the limbless mutation affects both processes. We present a model that explains the potential link between D-V positional information and apical ridge formation, and discuss the possible function of the limbless gene in terms of this model.

Reference

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
Chang
D. T.
,
Lopez
A.
,
von Kessler
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
Coelho
C. N. D.
,
Sumoy
L.
,
Rodgers
B. J.
,
Davidson
D. R.
,
Hill
R. E.
,
Upholt
W. B.
,
Kosher
R. A.
(
1991
)
Expression of the chicken homeobox-containing gene GHox-8 during embryonic chick limb development.
Mech. Dev
34
,
143
154
Coelho
C. N. D.
,
Krabbenhoft
K. M.
,
Upholt
W. B.
,
Fallon
J. F.
,
Kosher
R. A.
(
1991
)
Altered expression of the chicken homeobox-containing genes GHox-7 and Ghox-8 in the limb buds of limbless mutant chick embryos.
Development
113
,
1487
1293
Cohn
M. J.
,
Izpisúa-Belmonte
J. C.
,
Abud
H.
,
Heath
J. K.
,
Tickle
C.
(
1995
)
Fibroblast growth factors induce additional limb development from the flank of chick embryos.
Cell
80
,
739
746
Crossley
P. H.
,
Minowada
G.
,
MacArthur
C. A.
,
Martin
G. R.
(
1996
)
Roles for FGF8 in the induction, initiation, and maintenance of chick limb development.
Cell
84
,
127
136
Crossley
P. H.
,
Martinez
S.
,
Martin
G. R.
(
1996
)
Midbrain development induced by FGF8 in the chick embryo.
Nature
380
,
66
68
Davis
A. P.
,
Capecchi
M. R.
(
1994
)
Axial homeosis and appendicular skeleton defects in mice with a targeted disruption of hoxd-11.
Development
120
,
2187
2198
Davis
A. P.
,
Capecchi
M. R.
(
1996
)
A mutational analysis of the 5HoxD genes: dissection of genetic interactions during limb development in the mouse.
Development
122
,
1175
1185
Davis
C. A.
,
Holmyard
D. P.
,
Millen
K. J.
,
Joyner
A. L.
(
1991
)
Examining pattern formation in mouse, chicken and frog embryos with an En- specific antiserum.
Development
111
,
287
298
Dealy
C.N.
,
Kosher
R.A.
(
1996
)
IGF-I, insulin and Fgfs induce outgrowth of the limb buds of amelic mutant chick embryos.
Development
122
,
1323
1330
Dealy
C. N.
,
Roth
A.
,
Ferrari
D.
,
Brown
A. M. C.
,
Kosher
R. A.
(
1993
)
Wnt-5a and Wnt-7a are expressed in the developing chick limb bud in a manner suggesting roles in pattern formation along the proximodistal and dorsoventral axes.
Mech. Dev
43
,
175
186
Dolle
P.
,
Dierich
A.
,
LeMeur
M.
,
Schimmang
T.
,
Schuhbaur
N.
,
Chambon
P.
,
Duboule
D.
(
1993
)
Disruption of the Hoxd-13 gene induces localized heterochrony leading to mice with neotenic limbs.
Cell
75
,
431
441
Fallon
J. F.
,
Lopez
A.
,
Ros
M. A.
,
Savage
M. P.
,
Olwin
B. B.
,
Simandl
B. K.
(
1994
)
FGF-2: Apical ectodermal ridge growth signal for chick limb development.
Science
264
,
104
107
Favier
B.
,
LeMeur
M.
,
Chambon
P.
,
Dolle
P.
(
1995
)
Axial skeletal homeosis and forelimb malformations in Hoxd-11 mutant mice.
Proc. Natl. Acad. Sci. USA
92
,
310
314
Francis
P. H.
,
Richardson
M. K.
,
Brickell
P. M.
,
Tickle
C.
(
1994
)
Bone morphogenetic proteins and a signaling pathway that controls patterning in the developing chick limb.
Development
120
,
209
218
Francis-West
P. H.
,
Robertson
K. E.
,
Ede
D. A.
,
Rodriguez
C.
,
Izpisúa-Belmonte
J.-C.
,
Houston
B.
,
Burt
D. W.
,
Gribbin
C.
,
Brickell
P. M.
,
Tickle
C.
(
1995
)
Expression of genes encoding bone morphogenetic proteins and sonic hedgehog in talpid (ta3) limb buds: their relationships in the signalling cascade involved in limb patterning.
Dev. Dynamics
203
,
187
197
Gardner
C. A.
,
Barald
K. F.
(
1992
)
Expression patterns of Engrailed-like proteins inthe chick embryo.
Dev. Dynamics
193
,
370
388
Geduspan
J. S.
,
MacCabe
J. A.
(
1987
)
The ectodermal control of mesodermal patterns of differentiation in the developing chick wing.
Dev. Biol
124
,
398
408
Geduspan
J. S.
,
MacCabe
J. A.
(
1989
)
Transfer of dorsoventral information from mesoderm to ectoderm at the onset of limb development.
Anat. Rec
224
,
79
87
Geduspan
J. S.
,
Solursh
M.
(
1992
)
A growth-promoting influence from the mesonephros during limb outgrowth.
Dev. Biol
151
,
212
250
Hamburger
V.
,
Hamilton
H.
(
1951
)
A series of normal stages in the development of the chick embyros.
J. Morphol.
88
,
49
92
. Reprinted in. Dev. Dynamics 195, 231–272 (1992)
Houston
B.
,
Thorp
B. H.
,
Burt
D. W.
(
1994
)
Molecular cloning and expression of bone morphogenetic protein-7 in the chick epiphyseal growth plate.
J. Mol. Endocrin
13
,
289
301
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
Izpisúa-Belmonte
J.-C.
,
Duboule
D.
(
1992
)
Homeobox genes and pattern formation in the vertebrate limb.
Dev. Biol
152
,
26
36
Johnson
R. L.
,
Riddle
R. D.
,
Tabin
C.
(
1994
)
Mechanisms of limb patterning.
Curr. Opin. Genet. Dev
4
,
535
542
Laufer
E.
,
Nelson
C. E.
,
Johnson
R. I.
,
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
Logan
C.
,
Hanks
M.
,
Noble-Topham
S.
,
Nallainathan
D.
,
Provart
N.
,
Joyner
A.
(
1992
)
Cloning and sequence comparison of the mouse, human, and chicken engrailed genes reveal potential functional domains and regulatory regions.
Dev. Genet
13
,
345
358
Lopez-Martínez
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.
Curr. Biol
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
Martin
G. R.
(
1995
)
Why thumbs are up. News and Views.
Nature
374
,
410
411
Meinhardt
H.
(
1983
)
A boundary model for pattern formation in vertebrate limbs.
J. Embryol. Exp. Morph
76
,
115
137
Meinhardt
H.
(
1983
)
Cell determination boundaries as organizing regions for secondary embryonic fields.
Dev. Biol
96
,
375
385
Nelson
C. E.
,
Morgan
B. A.
,
Burke
A. C.
,
Laufer
E.
,
DiMambro
E.
,
Murtaugh
L. C.
,
Gonzales
E.
,
Tessarollo
L.
,
Parada
L. F.
,
Tabin
C.
(
1996
)
Analysis of Hox gene expression in the chick limb bud.
Development
122
,
1449
1466
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
Niswander
L.
,
Jeffrey
S.
,
Martin
G.
,
Tickle
C.
(
1994
)
Signaling in vertebrate limb development: a positive feedback loop between sonic hedgehog and FGF4.
Nature
371
,
609
612
Ohuchi
H.
,
Nakagawa
T.
,
Yamauchi
M.
,
Ohata
T.
,
Yoshioka
H.
,
Kuwana
T.
,
Mima
T.
,
Mikawa
T.
,
Nohno
T.
,
Noji
S.
(
1995
)
An additional limb can be induced from the flank of the chick embryo by FGF4.
Biochem. Biophys. Res. Comm
209
,
809
816
Parr
B. A.
,
McMahon
A. P.
(
1995
)
Dorsalizing signal Wnt-7a required for normal polarity of D-V and A-P axes of mouse limb.
Nature
374
,
350
353
Prahlad
K. V.
,
Skala
G.
,
Jones
D. G.
,
Briles
W. E.
(
1979
)
Limbless: A new genetic mutant in the chick.
J. Exp. Zool
209
,
427
434
Riddle
R. D.
,
Johnson
R. L.
,
Laufer
E.
,
Tabin
C.
(
1993
)
Sonic hedgehog mediates the polarizing activity of the ZPA.
Cell
75
,
1401
1416
Riddle
R. D.
,
Ensini
M.
,
Nelson
C.
,
Tsuchida
T.
,
Jessell
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
Robert
B.
,
Lyons
G.
,
Simandl
B. K.
,
Kuroiwa
A.
,
Buckingham
M.
(
1991
)
The apical ectodermal ridge regulates Hox-7 and Hox-8 gene expression in developing chick limb buds.
Genes Dev
5
,
2363
2374
Ros
M. A.
,
Lopez-Martínez
A.
,
Simandl
B. K.
,
Rodriguez
C.
,
Izpisúa-Belmonte
J.-C.
,
Dahn
R.
,
Fallon
J. F.
(
1996
)
The limb field mesoderm determines initial limb bud anteroposterior asymmetry and budding independent of sonic hedgehog or apical ectodermal gene expressions.
Development
122
,
2319
2330
Rowe
D. A.
,
Fallon
J. F.
(
1982
)
The proximodistal determination of skeletal parts in the developing chick leg.
J. Embryol. Exp. Morph
68
,
1
7
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
403
Saunders
J. W. J.
,
Reuss
C.
(
1974
)
Inductive and axial properties of prospective wing-bud mesoderm in the chick embryo.
Dev. Biol
38
,
41
50
Searls
R. L.
,
Zwilling
E.
(
1964
)
Regeneration of the apical ectodermal ridge of the chick limb bud.
Dev. Biol
9
,
38
55
Searls
R. L.
,
Janners
M. Y.
(
1971
)
The initiation of limb bud outgrowth in the embryonic chick.
Dev. Biol
24
,
198
213
Stephens
T. D.
,
McNulty
T. R.
(
1981
)
Evidence for a metameric pattern in the development of the chick humerus.
J. Embryol. Exp. Morph
61
,
191
205
Strecker
T. R.
,
Stephens
T. D.
(
1983
)
Peripheral nerves do not play a trophic role in limb skeletal morphogenesis.
Teratology
27
,
159
167
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
Summerbell
D.
,
Lewis
J. H.
,
Wolpert
L.
(
1973
)
Positional information in chick limb morphogenesis.
Nature
224
,
492
496
Tickle
C.
,
Eichele
G.
(
1994
)
Vertebrate limb development.
Annu. Rev. Cell Biol
10
,
121
152
Todt
W. L.
,
Fallon
J. F.
(
1984
)
Development of the apical ectodermal ridge in the chick wing bud.
J. Embryol. Exp. Morph
80
,
21
41
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.
,
Rodriguez
C.
,
Warnken
W.
,
Izpisúa-Belmonte
J.-C.
(
1995
)
Dorsal cell fate specified by chick Lmx1 during vertebrate limb development.
Nature
378
,
716
720
Vogel
A.
,
Rodriguez
C.
,
Izpisúa-Belmonte
J.-C.
(
1996
)
Involvement of FGF-8 in initiation, outgrowth and patterning of the vertebrate limb.
Development
122
,
1737
1750
Yang
Y.
,
Niswander
L.
(
1995
)
Interaction between the signaling molecules WNT7a and SHH during vertebrate limb development: dorsal signals regulate anteroposterior patterning.
Cell
80
,
939
947
Yokouchi
Y.
,
Ohsugi
K.
,
Sasaki
H.
,
Kuroiwa
A.
(
1991
)
Chicken homeobox gene Msx-1: structure, expression in limb buds and effect of retinoic acid.
Development
113
,
431
444
This content is only available via PDF.