In this study, we have analyzed the expression and function of Gremlin in the developing avian limb. Gremlin is a member of the DAN family of BMP antagonists highly conserved through evolution able to bind and block BMP2, BMP4 and BMP7. At early stages of development, gremlin is expressed in the dorsal and ventral mesoderm in a pattern complementary to that of bmp2, bmp4 and bmp7. The maintenance of gremlin expression at these stages is under the control of the AER, ZPA, and BMPs. Exogenous administration of recombinant Gremlin indicates that this protein is involved in the control of limb outgrowth. This function appears to be mediated by the neutralization of BMP function to maintain an active AER, to restrict the extension of the areas of programmed cell death and to confine chondrogenesis to the central core mesenchyme of the bud. At the stages of digit formation, gremlin is expressed in the proximal boundary of the interdigital mesoderm of the chick autopod. The anti-apoptotic influence of exogenous Gremlin, which results in the formation of soft tissue syndactyly in the chick, together with the expression of gremlin in the duck interdigital webs, indicates that Gremlin regulates the regression of the interdigital tissue. At later stages of limb development, gremlin is expressed in association with the differentiating skeletal pieces, muscles and the feather buds. The different expression of Gremlin in relation with other BMP antagonists present in the limb bud, such as Noggin, Chordin and Follistatin indicates that the functions of BMPs are regulated specifically by the different BMP antagonists, acting in a complementary fashion rather than being redundant signals.

Amthor
H.
,
Christ.
B.
,
Weil
M.
,
Patel
K.
(
1998
)
The importance of timing differentiation during limb muscle development.
Curr. Biol
8
,
642
652
Brunet
L. J.
,
MacMahon
J. A.
,
McMahon
A. P.
,
Harland
R. M.
(
1998
)
Noggin, cartilage morphogenesis, and joint formation in the mammalian skeleton.
Science
280
,
1455
1457
Capdevila
J.
,
Johnson
R.L.
(
1998
)
Endogenous and ectopic expression of noggin suggests a conserved mechanism for regulation of BMP function during limb and somite patterning.
Dev. Biol
197
,
205
217
Duprez
D. M.
,
Bell
E. J.
,
Richardson
M.
,
Archer
C. W.
,
Wolpert
L.
,
Brickell
P. M.
,
Francis-West
P. H.
(
1996
)
Overexpression of BMP-2 and BMP-4 alters the size and shape of developing skeletal elements in the chick limb.
Mech. Dev
57
,
145
157
Duprez
D.
,
Coltey
M.
,
Amthor
H.
,
Brickell
P. M.
,
Tickle
C.
(
1996
)
Bone morphogenetic protein-2 (BMP-2) inhibits muscle development and promotes cartilage formation in chick limb bud cultures.
Dev. Biol
174
,
448
452
Duprez
D. M.
,
Kostakopoulou
K.
,
Francis-West
P. H.
,
Tickle
C.
,
Brickell
P. M.
(
1996
)
Activation of expression of FGF-4 and HoxD gene expression by BMP-2 expressing cells in the developing chick limb.
Development
122
,
1821
1828
Enomoto-Iwamoto
M.
,
Iwamoto
M.
,
Mukudai
Y.
,
Kawakami
Y.
,
Nohno
T.
,
Higuchi
Y.
,
Takemoto
S.
,
Ohuchi
H.
,
Noji
S.
,
Kurisu
K.
(
1998
)
Bone morphogenetic protein signaling is required for maintenance of differentiation phenotype, control of proliferation, and hypertrophy in chondrocytes.
J. Cell Biol
140
,
409
418
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. E.
,
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 signaling cascade involved in limb patterning.
Dev. Dyn
203
,
187
197
Francis-West
P. H.
,
Parish
J.
,
Lee
K.
,
Archer
C.
(
1999
)
BMP/GDF-signalling interactions during synovial joint development.
Cell Tissue Res
296
,
111
119
Gañan
Y.
,
Macias
D.
,
Duterque-Coquillaud
M.
,
Ros
M. A.
,
Hurle
J. M.
(
1996
)
Role of TGFs and BMPs as signals controlling the position of the digits and the areas of interdigital cell death in the developing chick limb autopod.
Development
122
,
2349
2357
Gañan
Y.
,
Macias
D.
,
Basco
R.D.
,
Merino
R.
,
Hurle
J. M.
(
1998
)
Morphological diversity of the avian foot is related with the pattern of msx gene expression in the developing autopod.
Dev. Biol
196
,
33
41
Graham
A.
,
Francis-West
P.
,
Brickell
P.
,
Lumsden
A.
(
1994
)
The signalling molecule BMP4 mediates apotosis in the rhombencephalic neural crest.
Nature
372
,
684
686
Hamburger
V.
,
Hamilton
H. L.
(
1951
)
A series of normal stages in the development of the chick embryo.
J. Morphol
88
,
49
92
Healy
C.
,
Uwangoho
D.
,
Sharpe
P. T.
(
1999
)
Regulation and role of Sox9 in cartilage formation.
Dev. Dyn
215
,
69
78
Hofmann
C.
,
Drossopoulou
G.
,
McMahon
A.
,
Balling
R.
,
Tickle
C.
(
1998
)
Inhibitory action of BMPs on Pax1 expression and on shoulder girdle formation during limb development.
Dev. Dyn
213
,
199
206
Hogan
B. L. M.
(
1996
)
Bone morphogenetic proteins: Multifunctional regulators of vertebrate development.
Genes Dev
10
,
1580
1594
Hsu
D. R.
,
Economides
A. N.
,
Wang
X.
,
Eimon
P. M.
,
Harland
R.
(
1998
)
The Xenopus dorsalizing factor gremlin identifies a novel family of secreted proteins that antagonize BMP activities.
Molec. Cell
1
,
673
683
Jung
H.-S.
,
Francis-West
P. H.
,
Widelitz
R. B.
,
Jiang
T.-X.
,
Ting-Berreth
S.
,
Tickle
C.
,
Wolpert
L.
,
Chuong
C.-M.
(
1998
)
Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning.
Dev. Biol
196
,
11
23
Kawakami
Y.
,
Ishikawa
T.
,
Shimabara
M.
,
Tanda
N.
,
Enomoto-Iwamoto
M.
,
Iwamoto
M.
,
Kuwana
T.
,
Ueki
A.
,
Noji
S.
,
Nohno
T.
(
1996
)
BMP signaling during bone pattern determination in the developing limb.
Development
122
,
3557
3566
Laufer
E.
,
Pizette
S.
,
Zou
H.
,
Orozco
O. E.
,
Niswander
L.
(
1997
)
BMP expression in duck interdigital webbing: a reanalysis.
Science
278
,
305
–.
Li
S.
,
Anderson
R.
,
Reginelli
A. D.
,
Muneoka
K.
(
1996
)
FGF-2 influences cell movements and gene expression during limb development.
J. Exp. Zool
274
,
234
247
Lyons
K. M.
,
Hogan
B. L. M.
,
Robertson
E. J.
(
1995
)
Colocalization of BMP7 and BMP2 RNAs suggests that these factors cooperatively mediate tissue interactions during murine development.
Mech. Dev
50
,
71
83
Macias
D.
,
Gañan
Y.
,
Ros
M. A.
,
Hurle
J. M.
(
1996
)
In vivo inhibition of programmed cell death by local administration of FGF-2 and FGF-4 in the interdigital areas of the embryonic leg bud.
Anat. Embryol
193
,
533
541
Macias
D.
,
Gañan
Y.
,
Sampath
T. K.
,
Piedra
M. E.
,
Ros
M. A.
,
Hurle
J. M.
(
1997
)
Role of BMP-2 and OP-1 (BMP-7) in programmed cell death and skeletogenesis during chick limb development.
Development
124
,
1109
1117
Massague
J.
(
1996
)
TGFsignaling: Receptors, transducers and Mad proteins.
Cell
85
,
947
950
Merino
R.
,
Gañan
Y.
,
Macias
D.
,
Economides
A. N.
,
Sampath
K. T.
,
Hurle
J. M.
(
1998
)
Morphogenesis of digits in the avian limb is controlled by Fgfs, TGFs, and Noggin through BMP signaling.
Dev. Biol
200
,
35
45
Merino
R.
,
Macias
D.
,
Gañan
Y.
,
Rodriguez-Leon
J.
,
Economides
A.
,
Rodriguez-Esteban
C.
,
Izpisua-Belmonte
J. C.
,
Hurle
J. M.
(
1999
)
Control of digit formation by activin signalling.
Development
126
,
2161
2170
Niswander
L.
,
Martin
G.
(
1993
)
FGF-4 and BMP-2 have opposite effects on limb outgrowth.
Nature
361
,
68
71
Pearce
J. J. H.
,
Penny
G.
,
Rossant
J.
(
1999
)
A mouse Cerberus/Dan-related gene family.
Dev. Biol
209
,
98
110
Piccolo
S.
,
Sasai
Y.
,
Lu
B.
,
De Robertis
E. M.
(
1996
)
Dorsoventral patterning in Xenopus: Inhibition of ventral signals by direct binding of chordin to BMP-4.
Cell
86
,
589
598
Pizette
S.
,
Niswander
L.
(
1999
)
BMPs negatively regulate structure and function of the limb apical ectodermal ridge.
Development
126
,
883
894
Roark
E. R.
,
Greer
K.
(
1994
)
Transforming growth factor-and bone morphogenetic protein-2 act by distinct mechanisms to promote chick limb cartilage differentiation in vitro.
Dev. Dyn
200
,
103
116
Smith
W. C.
,
Harland
R. M.
(
1992
)
Expression cloning of noggin, a new dorsalizing factor localized to the Spemann organizer in Xenopus embryos.
Cell
70
,
829
840
Solursh
M.
(
1984
)
Ectoderm as a determinant of early tissue pattern in the limb bud.
Cell Differentiation
15
,
17
24
Stanley
E.
,
Biben
C.
,
Kotecha
S.
,
Fabri
L.
,
Tajbakhsh
S.
,
Wang
C.-C.
,
Hatzistavrou
T.
,
Roberts
B.
,
Drinkwater
C.
,
Lah
M.
,
Buckingham
M.
,
Hilton
D.
,
Nash
A.
,
Mohun
T.
,
Harvey
R.P.
(
1998
)
DAN is a secreted glycoprotein related to Xenopus cerberus.
Mech. Dev
77
,
173
184
Vogel
A.
,
Tickle
C.
(
1993
)
FGF-4 maintains polarizing activity of posterior limb bud cells in vivo and in vitro.
Development
119
,
199
206
Yokouchi
Y.
,
Sakiyama
J.
,
Kameda
T.
,
Iba
H.
,
Suzuki
A.
,
Ueno
N.
,
Kuroiwa
A.
(
1996
)
BMP-2/-4 mediate programmed cell death in chicken limb buds.
Development
122
,
3725
3734
Zimmerman
L. B.
,
De Jesús-Escobar
J. M.
,
Harland
R. M.
(
1996
)
The speman organizer signal noggin binds and inactivates bone morphogenetic protein 4.
Cell
86
,
599
606
Zou
H.
,
Niswander
L.
(
1996
)
Requirement for BMP signaling in interdigital apoptosis and scale formation.
Science
272
,
738
741
Zou
H.
,
Choe
K.-M.
,
Lu
Y.
,
Massague
J.
,
Niswander
L.
(
1997
)
BMP signaling and vertebrate limb development.
Cold. Spring Harbor Symp. Quant. Biol
62
,
269
272
Zou
H.
,
Wieser
R.
,
Massague
J.
,
Niswander
L.
(
1997
)
Distinct roles of type I bone morphogenetic protein receptors in the formation and differentiation of cartilage.
Genes Dev
11
,
2191
2203
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