Vertebrate limbs develop in a temporal proximodistal sequence, with proximal regions specified and generated earlier than distal ones. Whereas considerable information is available on the mechanisms promoting limb growth, those involved in determining the proximodistal identity of limb parts remain largely unknown. We show here that retinoic acid (RA) is an upstream activator of the proximal determinant genes Meis1 and Meis2. RA promotes proximalization of limb cells and endogenous RA signaling is required to maintain the proximal Meis domain in the limb. RA synthesis and signaling range, which initially span the entire lateral plate mesoderm, become restricted to proximal limb domains by the apical ectodermal ridge (AER) activity following limb initiation. We identify fibroblast growth factor (FGF) as the main molecule responsible for this AER activity and propose a model integrating the role of FGF in limb cell proliferation, with a specific function in promoting distalization through inhibition of RA production and signaling.

Abu-Shaar
M.
,
Mann
R. S.
(
1998
)
Generation of multiple antagonistic domains along the proximodistal axis during Drosophila leg development.
Development
125
,
3821
3830
Abu-Shaar
M.
,
Ryoo
H. D.
,
Mann
R. S.
(
1999
)
Control of the nuclearlocalization of extradenticle by competing nuclear import and export signals.
Genes Dev
13
,
935
945
Berggren
K.
,
McCaffery
P.
,
Drager
U.
,
Forehand
C. J.
(
1999
)
Differential distribution of retinoic acid synthesis in the chicken embryo as determined by immunolocalization of the retinoic acid synthetic enzyme, RALDH-2.
Dev. Biol
210
,
288
304
Berthelsen
J.
,
Kilstrup-Nielsen
C.
,
Blasi
F.
,
Mavilio
F.
,
Zappavigna
V.
(
1999
)
The subcellular localization of PBX1 and EXD proteins depends on nuclear import and export signals and is modulated by association with PREP1 and HTH.
Genes Dev
13
,
946
953
Capdevila
J.
,
Tsukui
T.
,
Rodriguez Esteban
C.
,
Zappavigna
V.
,
Izpisua Belmonte
J. C.
(
1999
)
Control of vertebrate limb outgrowth by the proximal factor Meis2 and distal antagonism of BMPs by Gremlin.
Mol. Cell
4
,
839
849
Cecconi
F.
,
Proetzel
G.
,
Alvarez-Bolado
G.
,
Jay
D.
,
Gruss
P.
(
1997
)
Expression of Meis2, a Knotted-related murine homeobox gene, indicates a role in the differentiation of the forebrain and the somitic mesoderm.
Dev. Dyn
210
,
184
190
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
Cohn
M. J.
,
Tickle
C.
(
1996
)
Limbs: a model for pattern formation within the vertebrate body plan.
Trends Genet
12
,
253
257
Crawford
K.
,
Stocum
D. L.
(
1988
)
Retinoic acid coordinately proximalizes regenerate pattern and blastema differential affinity in axolotl limbs.
Development
102
,
687
698
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
de The
H.
,
Vivanco-Ruiz
M. M.
,
Tiollais
P.
,
Stunnenberg
H.
,
Dejean
A.
(
1990
)
Identification of a retinoic acid responsive element in the retinoic acid receptor beta gene.
Nature
343
,
177
180
Dolle
P.
,
Ruberte
E.
,
Leroy
P.
,
Morriss-Kay
G.
,
Chambon
P.
(
1990
)
Retinoic acid receptors and cellular retinoid binding proteins. I. A systematic study of their differential pattern of transcription during mouse organogenesis.
Development
110
,
1133
1151
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
Gardiner
D. M.
,
Blumberg
B.
,
Komine
Y.
,
Bryant
S. V.
(
1995
)
Regulation of HoxA expression in developing and regenerating axolotl limbs.
Development
121
,
1731
1741
González-Crespo
S.
,
Abu-Shaar
M.
,
Torres
M.
,
Martínez-A
C.
,
Mann
R. S.
,
Morata
G.
(
1998
)
Antagonism between extradenticle function and Hedgehog signalling in the developing limb.
Nature
394
,
196
200
González-Crespo
S.
,
Morata
G.
(
1996
)
Genetic evidence for the subdivision of the arthropod limb into coxopodite and telopodite.
Development
122
,
3921
3928
Helms
J.
,
Thaller
C.
,
Eichele
G.
(
1994
)
Relationship between retinoic acid and sonic hedgehog, two polarizing signals in the chick wing bud.
Development
120
,
3267
3274
Helms
J. A.
,
Kim
C. H.
,
Eichele
G.
,
Thaller
C.
(
1996
)
Retinoic acid signaling is required during early chick limb development.
Development
122
,
1385
1394
Ide
H.
,
Wada
N.
,
Uchiyama
K.
(
1994
)
Sorting out of cells from different parts and stages of the chick limb bud.
Dev. Biol
162
,
71
76
Irving
C.
,
Mason
I.
(
2000
)
Signalling by FGF8 from the isthmus patterns anterior hindbrain and establishes the anterior limit of Hox gene expression.
Development
127
,
177
186
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
Johnson
R. L.
,
Tabin
C. J.
(
1997
)
Molecular models for vertebrate limb development.
Cell
90
,
979
990
Kamps
M. P.
,
Murre
C.
,
Sun
X.
,
Baltimore
D.
(
1990
)
A new homeobox gene contributes the DNA binding domain of the t(1;19) translocation protein in pre-B ALL.
Cell
60
,
547
555
Kim
W. S.
,
Stocum
D. L.
(
1986
)
Retinoic acid modifies positional memory in the anteroposterior axis of regenerating axolotl limbs.
Dev. Biol
114
,
170
179
Kochhar
D. M.
,
Jiang
H.
,
Penner
J. D.
,
Johnson
A. T.
,
Chandraratna
R. A.
(
1998
)
The use of a retinoid receptor antagonist in a new model to study vitamin A-dependent developmental events.
Int. J. Dev. Biol
42
,
601
608
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
Logan
M.
,
Pagan-Westphal
S. M.
,
Smith
D. M.
,
Paganessi
L.
,
Tabin
C. J.
(
1998
)
The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals.
Cell
94
,
307
317
Logan
M.
,
Tabin
C.
(
1998
)
Targeted gene misexpression in chick limb buds using avian replication-competent retroviruses.
Methods
14
,
407
420
Maden
M.
(
1982
)
Vitamin A and pattern formation in the regenerating limb.
Nature
295
,
672
675
Maden
M.
(
1993
)
The homeotic transformation of tails into limbs in Rana temporaria by retinoids.
Dev. Biol
159
,
379
391
Mendelsohn
C.
,
Ruberte
E.
,
Chambon
P.
(
1992
)
Retinoid receptors in vertebrate limb development.
Dev. Biol
152
,
50
61
Mendelsohn
C.
,
Ruberte
E.
,
LeMeur
M.
,
Morriss-Kay
G.
,
Chambon
P.
(
1992
)
Developmental analysis of the retinoic acid-inducible RAR-beta 2 promoter in transgenic animals.
Development
113
,
723
734
Mercader
N.
,
Leonardo
E.
,
Azpiazu
N.
,
Serrano
A.
,
Morata
G.
,
Martinez
C.
,
Torres
M.
(
1999
)
Conserved regulation of proximodistal limb axis development by Meis1/Hth.
Nature
402
,
425
429
Mohammadi
M.
,
McMahon
G.
,
Sun
L.
,
Tang
C.
,
Hirth
P.
,
Yeh
B. K.
,
Hubbard
S. R.
,
Schlessinger
J.
(
1997
)
Structures of the tyrosine kinase domain of fibroblast growth factor receptor in complex with inhibitors.
Science
276
,
955
960
Mohanty-Hejmadi
P.
,
Dutta
S. K.
,
Mahapatra
P.
(
1992
)
Limbs generated at site of tail amputation in marbled balloon frog after vitamin A treatment.
Nature
355
,
352
353
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
Moskow
J. J.
,
Bullrich
F.
,
Huebner
K.
,
Daar
I. O.
,
Buchberg
A. M.
(
1995
)
Meis1, a PBX1-related homeobox gene involved in myeloid leukemia in BXH-2 mice.
Mol. Cell Biol
15
,
5434
5443
Nakamura
T.
,
Jenkins
N. A.
,
Copeland
N. G.
(
1996
)
Identification of a new family of Pbx-related homeobox genes.
Oncogene
13
,
2235
2242
Niederreither
K.
,
Subbarayan
V.
,
Dolle
P.
,
Chambon
P.
(
1999
)
Embryonic retinoic acid synthesis is essential for early mouse post-implantation development.
Nat. Genet
21
,
444
448
Niederreither
K.
,
Ward
S. J.
,
Dolle
P.
,
Chambon
P.
(
1996
)
Morphological and molecular characterization of retinoic acid-induced limb duplications in mice.
Dev. Biol
176
,
185
198
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
Nourse
J.
,
Mellentin
J. D.
,
Galili
N.
,
Wilkinson
J.
,
Stanbridge
E.
,
Smith
S. D.
,
Cleary
M. L.
(
1990
)
Chromosomal translocation t(1;19) results in synthesis of a homeobox fusion mRNA that codes for a potential chimeric transcription factor.
Cell
60
,
535
545
Oulad-Abdelghani
M.
,
Chazaud
C.
,
Bouillet
P.
,
Sapin
V.
,
Chambon
P.
,
Dolle
P.
(
1997
)
Meis2, a novel mouse Pbx-related homeobox gene induced by retinoic acid during differentiation of P19 embryonal carcinoma cells.
Dev. Dyn
210
,
173
183
Pecorino
L. T.
,
Entwistle
A.
,
Brockes
J. P.
(
1996
)
Activation of a single retinoic acid receptor isoform mediates proximodistal respecification.
Curr. Biol
6
,
563
569
Rauskolb
C.
,
Peifer
M.
,
Wieschaus
E.
(
1993
)
extradenticle, a regulator of homeotic gene activity, is a homolog of the homeobox-containing human proto-oncogene pbx1.
Cell
74
,
1101
1112
Rieckhof
G. E.
,
Casares
F.
,
Ryoo
H. D.
,
Abu-Shaar
M.
,
Mann
R. S.
(
1997
)
Nuclear translocation of extradenticle requires homothorax, which encodes an extradenticle-related homeodomain protein.
Cell
91
,
171
183
Rossant
J.
,
Zirngibl
R.
,
Cado
D.
,
Shago
M.
,
Giguere
V.
(
1991
)
Expression of a retinoic acid response element-hsplacZ transgene defines specific domains of transcriptional activity during mouse embryogenesis.
Genes Dev
5
,
1333
1344
Rutledge
J. C.
,
Shourbaji
A. G.
,
Hughes
L. A.
,
Polifka
J. E.
,
Cruz
Y. P.
,
Bishop
J. B.
,
Generoso
W. M.
(
1994
)
Limb and lower-body duplications induced by retinoic acid in mice.
Proc. Natl Acad. Sci. USA
91
,
5436
5440
Saunders
J. W.
(
1948
)
The proximodistal sequence of origin of the parts of the chick wing and the role of the ectoderm.
J. Exp. Zool
108
,
363
403
Schneider
A.
,
Mijalski
T.
,
Schlange
T.
,
Dai
W.
,
Overbeek
P.
,
Arnold
H. H.
,
Brand
T.
(
1999
).
The homeobox gene NKX3.2 is a target of left-right signalling and is expressed on opposite sides in chick and mouse embryos.
Curr. Biol
9
,
911
914
Schwabe
J. W.
,
Rodriguez-Esteban
C.
,
Izpisúa Belmonte
J. C.
(
1998
)
Limbs are moving: where are they going?.
Trends Genet
14
,
229
235
Simon
H. G.
,
Tabin
C. J.
(
1993
).
Analysis of Hox-4.5 and Hox-3.6 expression during newt limb regeneration: differential regulation of paralogous Hox genes suggest different roles for members of different Hox clusters.
Development
117
,
1397
1407
Stratford
T.
,
Horton
C.
,
Maden
M.
(
1997
)
Retinoic acid is required for the initiation of outgrowth in the chick limb bud.
Curr. Biol
6
,
1124
1133
Sucov
H. M.
,
Murakami
K. K.
,
Evans
R. M.
(
1990
)
Characterization of an autoregulated response element in the mouse retinoic acid receptor type beta gene.
Proc. Natl Acad. Sci. USA
87
,
5392
5396
Summerbell
D.
(
1983
)
The effect of local application of retinoic acid to the anterior margin of the developing chick limb bud.
J. Embryol. Exp. Morph
78
,
269
289
Summerbell
D.
,
Lewis
J.
,
Wolpert
L.
(
1973
)
Positional information in chick limb morphogenesis.
Nature
224
,
492
496
Swindell
E. C.
,
Thaller
C.
,
Sockanathan
S.
,
Petkovich
M.
,
Jessell
T. M.
,
Eichele
G.
(
1999
)
Complementary domains of retinoic acid production and degradation in the early chick embryo.
Dev. Biol
216
,
282
296
Tamura
K.
,
Yokouchi
Y.
,
Kuroiwa
A.
,
Ide
H.
(
1997
)
Retinoic acid changes the proximodistal developmental competence and affinity of distal cells in the developing chick limb bud.
Dev. Biol
188
,
224
234
Tickle
C.
,
Alberts
B.
,
Wolpert
L.
,
Lee
J.
(
1982
)
Local application of retinoic acid to the limb bond mimics the action of the polarizing region.
Nature
296
,
564
566
Tickle
C.
,
Crawley
A.
,
Farrar
J.
(
1989
)
Retinoic acid application to chick wing buds leads to a dose-dependent reorganization of the apical ectodermal ridge that is mediated by the mesenchyme.
Development
106
,
691
705
Tickle
C.
,
Eichele
G.
(
1994
)
Vertebrate limb development.
Annu. Rev. Cell Biol
10
,
121
152
Tickle
C.
,
Lee
J.
,
Eichele
G.
(
1985
)
A quantitative analysis of the effect of all-trans-retinoic acid on the pattern of chick wing development.
Dev. Biol
109
,
82
95
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
Wilkinson
D. G.
,
Nieto
M. A.
(
1993
)
Detection of messenger RNA by in situ hybridization to tissue sections and whole mount.
Methods Enzymol
225
,
361
373
This content is only available via PDF.