Patterning of the vertebrate limb along the anterior-posterior axis is controlled by the zone of polarizing activity (ZPA) located at the posterior limb margin. One of the vertebrate Hh family members, Shh, has been shown to be able to mediate the function of the ZPA. Several naturally occurring mouse mutations with the phenotype of preaxial polydactyly exhibit ectopic Shh expression at the anterior limb margin. In this study, we report the molecular characterization of a spontaneous mouse mutation, Doublefoot (Dbf). Dbf is a dominant mutation which maps to chromosome 1. Heterozygous and homozygous embryos display a severe polydactyly with 6 to 8 digits on each limb. We show here that Shh is expressed normally in Dbf mutants. In contrast, a second Hh family member, Indian hedgehog (Ihh) which maps close to Dbf, is ectopically expressed in the distal limb bud. Ectopic Ihh expression in the distal and anterior limb bud results in the ectopic activation of several genes associated with anterior-posterior and proximal-distal patterning (Fgf4, Hoxd13, Bmp2). In addition, specific components in the Hedgehog pathway are either ectopically activated (Ptc, Ptc-2, Gli1) or repressed (Gli2). We propose that misexpression of Ihh, and not a novel Smoothened ligand as recently suggested (Hayes et al., 1998), is responsible for the Dbf phenotype. We consider that Ihh has a similar activity to Shh when expressed in the early Shh-responsive limb bud. To determine whether Dbf maps to the Ihh locus, which is also on chromosome 1, we performed an interspecific backcross. These results demonstrate that Dbf and Ihh are genetically separated by approximately 1.3 centimorgans, suggesting that Dbf mutation may cause an exceptionally long-range disruption of Ihh regulation. Although this leads to ectopic activation of Ihh, normal expression of Ihh in the cartilaginous elements is retained.

REFERENCES

Bedell
M. A.
,
Brannan
C. I.
,
Evans
E. P.
,
Copela
N. G.
,
Jenkins
N. A.
,
Donovan
P.J.
(
1995
)
DNA rearrangements located over 100 kb 5of the Steel (Sl)-coding region in Steel-panda and Steel-contrasted mice deregulate Sl expression and cause female sterility by disrupting ovarian follicle development.
Genes Dev
9
,
455
470
Bitgood
M. J.
,
McMahon
A. P.
(
1995
)
Hedgehog and Bmp genes are coexpressed at many diverse sites of cell-cell interaction in the mouse embryo.
Dev. Biol
172
,
126
138
Buscher
D.
,
Bosse
B.
,
Heymer
J.
,
Ruther
U.
(
1997
)
Evidence for genetic control of Sonic hedgehog by Gli3 in mouse limb development.
Mech. Dev
62
,
175
182
Buscher
D.
,
Ruther
U.
(
1998
)
Expression profile of Gli family members and Shh in normal and mutant mouse limb development.
Dev. Dyn
211
,
88
96
Chan
D. C.
,
Laufer
E.
,
Tabin
C.
,
Leder
P.
(
1995
)
Polydactylous limbs in Strong's Luxoid mice result from ectopic polarizing activity.
Development
121
,
1971
1978
Charite
J.
,
de Graaff
W.
,
Shen
S.
,
Deschamps
J.
(
1994
)
Ectopic expression of Hoxb-8 causes duplication of the ZPA in the forelimb and homeotic transformation of axial structures.
Cell
78
,
589
601
Cooke
J.
,
Summerbell
D.
(
1981
)
Control of growth related to pattern specification in chick wing-bud mesenchyme.
J. Embryol. Exp. Morphol
65
,
169
185
Crossley
P.
,
Martin
G.
(
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
Cygan
J.
,
Johnson
R.
,
McMahon
A.
(
1997
)
Novel regulatory interactions revealed by studies of murine limb pattern in Wnt-7a and En-1 mutants.
Development
124
,
5021
5032
Dolle
P.
,
Izpisua-Belmonte
J. C.
,
Boncinelli
E.
,
Duboule
D.
(
1991
).
The Hox-4.8 gene is localized at the 5extremity of the Hox-4 complex and is expressed in the most posterior parts of the body during development.
Mech, Dev
36
,
3
13
Dominguez
M.
,
Brunner
M.
,
Hafen
E.
,
Basler
K.
(
1996
)
Sending and receiving the hedgehog signal: control by the Drosophila Gli protein Cubitus interruptus.
Science
272
,
1621
1625
Echelard
Y.
,
Epstein
D. J.
,
St-Jacques
B.
,
Shen
L.
,
Mohler
J.
,
McMahon
J. A.
,
McMahon
A. P.
(
1993
)
Sonic hedgehog, a memberof a family of putative signaling molecules, is implicated in the regulation of CNS polarity.
Cell
75
,
1417
1430
Ericson
J.
,
Muhr
J.
,
Placzek
M.
,
Lints
T.
,
Jessell
T. M.
,
Edlund
T.
(
1995
)
Sonic hedgehog induces the differentiation of ventral forebrain neurons: a common signal for ventral patterning within the neural tube.
Cell
81
,
747
756
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
Fan
C. M.
,
Porter
J. A.
,
Chiang
C.
,
Chang
D. T.
,
Beachy
P. A.
,
Tessier-Lavigne
M.
(
1995
)
Long-range sclerotome induction by sonic hedgehog: direct role of the amino-terminal cleavage product and modulation by the cyclic AMP signaling pathway.
Cell
81
,
457
465
Ganan
Y.
,
Macias
D.
,
Duterque-Coquillaud
M.
,
Ros
M. A.
,
Hurle
J. M.
(
1996
)
Role of TGF beta s 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
Goodrich
L. V.
,
Johnson
R. L.
,
Milenkovic
L.
,
McMahon
J. A.
,
Scott
M. P.
(
1996
)
Conservation of the hedgehog/patched signaling pathway from flies to mice: induction of a mouse patched gene by Hedgehog.
Genes Dev
10
,
301
312
Goulding
M. D.
,
Chalepakis
G.
,
Deutsch
U.
,
Erselius
J. R.
,
Gruss
P.
(
1991
)
Pax-3, a novel murine DNA binding protein expressed during early neurogenesis.
EMBO J
10
,
1135
1147
Hayes
C.
,
Brown
J. M.
,
Lyon
M. F.
,
Morriss-Kay
G. M.
(
1998
)
Sonic hedgehog is not required for polarizing activity in the Doublefoot mutant mouse limb bud.
Development
125
,
351
357
Hui
C. C.
,
Joyner
A. L.
(
1993
)
A mouse model of greig cephalopolysyndactyly syndrome: the extra-toes mutation contains an intragenic deletion of the Gli3 gene.
Nat. Genet
3
,
241
246
Johnson
R. L.
,
Tabin
C. J.
(
1997
)
Molecular models for vertebrate limb development.
Cell
90
,
979
990
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
Loomis
C.
,
Kimmel
R.
,
Tong
C. X.
,
Michaud
J.
,
Joyner
A.
(
1998
)
Analysis of the genetic pathway leading to formation of ectopic apical ectodermal ridges in mouse Engrailed-1 mutant limbs.
Development
125
,
1137
1148
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.
Curr. Biol
5
,
791
796
Lyon
M. F.
,
Quinney
R.
,
Glenister
P. H.
,
Kerscher
S.
,
Guillot
P.
,
Boyd
Y.
(
1996
)
Doublefoot: a new mouse mutant affecting development of limbs and head.
Genet. Res
68
,
221
231
Lyons
K. M.
,
Pelton
R. W.
,
Hogan
B. L.
(
1989
)
Patterns of expression of murine Vgr-1 and BMP-2a RNA suggest that transforming growth factor-beta-like genes coordinately regulate aspects of embryonic development.
Genes Dev
3
,
1657
1668
Marigo
V.
,
Roberts
D. J.
,
Lee
S. M.
,
Tsukurov
O.
,
Levi
T.
,
Gastier
J. M.
,
Epstein
D. J.
,
Gilbert
D. J.
,
Copeland
N. G.
,
Seidman
C. E.
(
1995
)
Cloning, expression, and chromosomal location of SHH and IHH: two human homologues of the Drosophila segment polarity gene hedgehog.
Genomics
28
,
44
51
Marigo
V.
,
Scott
M. P.
,
Johnson
R. L.
,
Goodrich
L. V.
,
Tabin
C. J.
(
1996
)
Conservation in hedgehog signaling: induction of a chicken patched homolog by Sonic hedgehog in the developing limb.
Development
122
,
1225
1233
Marigo
V.
,
Tabin
C. J.
(
1996
)
Regulation of patched by sonic hedgehog in the developing neural tube.
Proc. Natl. Acad. Sci. USA
93
,
9346
9351
Marti
E.
,
Bumcrot
D. A.
,
Takada
R.
,
McMahon
A. P.
(
1995
)
Requirement of 19K form of Sonic hedgehog for induction of distinct ventral cell types in CNS explants.
Nature
375
,
322
325
Masuya
H.
,
Sagai
T.
,
Moriwaki
K.
,
Shiroishi
T.
(
1997
)
Multigenic control of the localization of the zone of polarizing activity in limb morphogenesis in the mouse.
Dev. Biol
182
,
42
51
Masuya
H.
,
Sagai
T.
,
Wakana
S.
,
Moriwaki
K.
,
Shiroishi
T.
(
1995
)
A duplicated zone of polarizing activity in polydactylous mouse mutants.
Genes Dev
9
,
1645
1653
Milaire
J.
,
Goffard
J. C.
(
1995
)
Histological analysis of limb defectsinduced in developing limb buds of NMRI mouse embryos after oral administration of 3–3-dimethyl-1-phenyltriazene (DMPT) to their mother on day 10 of gestation.
Eur. J. Morphol
33
,
491
508
Mo
R.
,
Freer
A. M.
,
Zinyk
D. L.
,
Crackower
M. A.
,
Michaud
J.
,
Heng
H. H.
,
Chik
K. W.
,
Shi
X. M.
,
Tsui
L. C.
,
Cheng
S. H.
,
Joyner
A. L.
,
Hui
C.
(
1997
)
Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development.
Development
124
,
113
123
Motoyama
J.
,
Takabatake
T.
,
Takeshima
K.
,
Hui
C.
(
1998
)
Ptch2, a second mouse Patched gene is co-expressed with Sonic hedgehog.
Nat. Genet
18
,
104
106
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.
,
Jeffrey
S.
,
Martin
G. R.
,
Tickle
C.
(
1994
)
A positive feedback loop coordinates growth and patterning in the vertebrate limb.
Nature
371
,
609
612
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
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
Qu
S.
,
Niswender
K. D.
,
Ji
Q.
,
van der Meer
R.
,
Keeney
D.
,
Magnuson
M. A.
,
Wisdom
R.
(
1997
)
Polydactyly and ectopic ZPA formation in Alx-4 mutant mice.
Development
124
,
3999
4008
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
Riddle
R. D.
,
Johnson
R. L.
,
Laufer
E.
,
Tabin
C.
(
1993
)
Sonic hedgehog mediates the polarizing activity of the ZPA.
Cell
75
,
1401
1416
Roelink
H.
,
Porter
J. A.
,
Chiang
C.
,
Tanabe
Y.
,
Chang
D. T.
,
Beachy
P. A.
,
Jessell
T. M.
(
1995
)
Floor plate and motor neuron induction by different concentrations of the amino-terminal cleavage product of sonic hedgehog autoproteolysis.
Cell
81
,
445
455
Stone
D. M.
,
Hynes
M.
,
Armanini
M.
,
Swanson
T. A.
,
Gu
Q.
,
Johnson
R. L.
,
Scott
M. P.
,
Pennica
D.
,
Goddard
A.
,
Phillips
H.
,
Noll
M.
,
Hooper
J. E.
,
de Sauvage
F.
,
Rosenthal
A.
(
1996
)
The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog.
Nature
384
,
129
134
Vortkamp
A.
,
Franz
T.
,
Gessler
M.
,
Grzeschik
K. H.
(
1992
)
Deletion of GLI3 supports the homology of the human Greig cephalopolysyndactyly syndrome (GCPS) and the mouse mutant extra toes (Xt).
Mamm. Genome
3
,
461
463
Vortkamp
A.
,
Lee
K.
,
Lanske
B.
,
Segre
G. V.
,
Kronenberg
H. M.
,
Tabin
C. J.
(
1996
)
Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein.
Science
273
,
613
622
Wilkinson
D. G.
,
Nieto
M. A.
(
1993
)
Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts.
Methods Enzymol
225
,
361
373
Yang
Y.
,
Drossopoulou
G.
,
Chuang
P. T.
,
Duprez
D.
,
Marti
E.
,
Bumcrot
D.
,
Vargesson
N.
,
Clarke
J.
,
Niswander
L.
,
McMahon
A.
,
Tickle
C.
(
1997
)
Relationship between dose, distance and time in Sonic Hedgehog-mediated regulation of anteroposterior polarity in the chick limb.
Development
124
,
4393
4404
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
Zou
H.
,
Niswander
L.
(
1996
)
Requirement for BMP signaling in interdigital apoptosis and scale formation.
Science
272
,
738
741
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