Several 5′ members of the Hoxd cluster are expressed in nested posterior-distal domains of the limb bud suggesting a role in regulating anteroposterior pattern of skeletal elements. While loss-of-function mutants have demonstrated a regulatory role for these genes in the developing limb, extensive functional overlaps between various different Hox genes has hampered elucidation of the roles played by individual members. In particular, the function of Hoxd-12 in the limb remains obscure. Using a gain-of-function approach, we find that Hoxd-12 misexpression in transgenic mice produces apparent transformations of anterior digits to posterior morphology and digit duplications, while associated tibial hemimelia and other changes indicate that formation/growth of certain skeletal elements is selectively inhibited. If the digital arch represents an anterior bending of the main limb axis, then the results are all reconcilable with a model in which Hoxd-12 promotes formation of postaxial chondrogenic condensations branching from this main axis (including the anteriormost digit) and selectively antagonizes formation of ‘true’ preaxial condensations that branch from this main axis (such as the tibia). Hoxd-12 misexpression can also induce ectopic Sonic hedgehog (Shh) expression, resulting in mirror-image polydactyly in the limb. Misexpression of Hoxd-12 in other lateral plate derivatives (sternum, pelvis) likewise phenocopies several luxoid/luxate class mouse mutants that all share ectopic Shh signalling. This suggests that feedback activation of Shh expression may be a major function of Hoxd-12. Hoxd-12 can bind to and transactivate the Shh promoter in vitro. Furthermore, expression of either exogenous Hoxd-11 or Hoxd-12 in cultured limb bud cells, together with FGF, induces expression of the endogenous Shh gene. Together these results suggest that certain 5′ Hoxd genes directly amplify the posterior Shh polarizing signal in a reinforcing positive feedback loop during limb bud outgrowth.

REFERENCES

Becker
D.
,
Jiang
Z.
,
Knodler
P.
,
Deinard
A. S.
,
Eid
R.
,
Kidd
K. K.
,
Shashikant
C. S.
,
Ruddle
F. H.
,
Schughart
K.
(
1996
)
Conserved regulatory element involved in the early onset of Hoxb6 gene expression.
Dev. Dynamics
205
,
73
81
Benson
G. V.
,
Nguyen
T. H.
,
Maas
R. L.
(
1995
)
The expression pattern of the murine Hoxa-10 gene and the sequence recognition of its homeodomain reveal specific properties of Abdominal B -like genes.
Mol. Cell Biol
15
,
1591
1601
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
Carter
T. C.
(
1951
)
The genetics of luxate mice. I. Morphological abnormalities of heterozygotes and homozygotes.
J. Genetics
50
,
277
299
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
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
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
Chevallier
A.
(
1977
)
Origine des ceintures scapulaaires et pelviennes chez l'embryon d'oiseau.
J. Embryol. Exp. Morph
42
,
275
292
Cohn
M. J.
,
Tickle
C.
(
1996
)
Limbs: a model for pattern formation within the vertebrate body plan.
Trends Genet
12
,
253
257
Conlon
R. A.
,
Rossant
J.
(
1992
)
Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox 2 genes in vivo.
Development
116
,
357
368
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 5 HoxD genes: dissection of genetic interactions during limb development in the mouse.
Development
122
,
1175
1185
Davis
A. P.
,
Witte
D. P.
,
Hsieh-Li
H. M.
,
Potter
S. S.
,
Capecchi
M. R.
(
1995
)
Absence of radius and ulna in mice lacking hoxa-11 and hoxd-11.
Nature
375
,
791
795
Dolle
P.
,
Dierich
A.
,
LeMeur
M.
,
Schimmang
T.
,
Schuhbauer
B.
,
Chambon
P.
,
Duboule
D.
(
1993
)
Disruption of the Hoxd-13 gene induces localized heterochrony leading to mice with neotenic limbs.
Cell
75
,
431
441
Duboule
D.
(
1994
)
How to make a limb?.
Science
266
,
575
576
Duboule
D.
(
1995
)
Vertebrate Hox genes and proliferation: an alternative pathway to homeosis?.
Curr. Opin. Genet. Dev
5
,
525
528
Eid
R.
,
Koseki
H.
,
Schughart
K.
(
1993
)
Analysis of LacZ reporter genes in transgenic embryos suggests the presence of several cis-acting regulatory elements in the murine Hoxb-6 gene.
Dev. Dynamics
196
,
205
216
Ekker
S. C.
,
Jackson
D. G.
,
von Kessler
D. P.
,
Sun
B. I.
,
Young
K. E.
,
Beachy
P. A.
(
1994
)
The degree of variation in DNA sequence recognition among four Drosophila homeotic proteins.
EMBO J
13
,
3551
3560
Favier
B.
,
Le Meur
M.
,
Chambon
P.
,
Dolle
P.
(
1995
)
Axial skeleton homeosis and forelimb malformations in Hoxd-11 mutant mice.
Proc. Natl Acad. Sci. USA
92
,
310
314
Favier
B.
,
Rijli
F. M.
,
Fromental-Ramain
C.
,
Fraulob
V.
,
Chambon
P.
,
Dolle
P.
(
1996
)
Functional cooperation between the non-paralogous genes Hoxa-10 and Hoxd-11 in the developing forelimb and axial skeleton.
Development
122
,
449
460
Forsthoefel
P. F.
(
1962
)
Genetics and manifold effects of Strong's luxoid gene in the mouse, including its interactions with Green's luxoid and Carter's luxate genes.
J. Morphol
110
,
391
420
Forsthoefel
P. F.
(
1963
)
Observations on the sequence of blastemal condensations in the limbs of the mouse embryo.
Anat. Record
147
,
129
138
Fromental-Ramain
C.
,
Warot
X.
,
Messadecq
N.
,
Lemeur
M.
,
Dolle
P.
,
Chambon
P.
(
1996
)
Hoxa-13 and Hoxd-13 play a crucial role in the patterning of the limb autopod.
Development
122
,
2997
3011
Goff
D. J.
,
Tabin
C. J.
(
1997
)
Analysis of Hoxd-13 and Hoxd-11 misexpression in chick limb buds reveals that Hox genes affect both bone condensation and growth.
Development
124
,
627
636
Grieshammer
U.
,
Minowada
G.
,
Pisenti
J. M.
,
Abbott
U. K.
,
Martin
G. R.
(
1996
)
The chick limbless mutation causes abnormalities in limb bud dorsal-ventral patterning: implications for the mechanism of apical ridge formation.
Development
122
,
3851
3861
Hardy
A.
,
Richardson
M. K.
,
Francis-West
P. H.
,
Rodriguez
C.
,
Izpisua-Belmonte
J. C.
,
Duprez
D.
,
Wolpert
L.
(
1995
)
Gene expression, polarising activity and skeletal patterning in reaggregated hind limb mesenchyme.
Development
121
,
4329
4337
Hughes
S. H.
,
Greenhouse
J. J.
,
Petropoulos
C. J.
,
Sutrave
P.
(
1987
)
Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors.
J. Virol
61
,
3004
3012
Hui
C. C.
,
Joyner
A. L.
(
1993
)
A mouse model of Greig cephalopolysyndactyly syndrome: the extra-toes Jmutation contains an intragenic deletion of the Gli3 gene.
Nat. Genet
3
,
241
246
Izpisua-Belmonte
J. C.
,
Falkenstein
H.
,
Dolle
P.
,
Renucci
A.
,
Duboule
D.
(
1991
)
Murine genes related to the Drosophila AbdB homeotic genes are sequentially expressed during development of the posterior part of the body.
EMBO J
10
,
2279
89
Izpisua-Belmonte
J. C.
,
Duboule
D.
(
1992
)
Homeobox genes and pattern formation in the vertebrate limb.
Dev. Biol
152
,
26
36
Johnson
D. R.
(
1967
)
Extra-toes: a new mutant gene causing multiple abnormalities in the mouse.
J. Embryol. Exp. Morph
17
,
543
581
Kessel
M.
,
Gruss
P.
(
1991
)
Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid.
Cell
67
,
89
104
Kondo
T.
,
Dolle
P.
,
Zakany
J.
,
Duboule
D.
(
1996
)
Function of posterior HoxD genes in the morphogenesis of the anal sphincter.
Development
122
,
2651
2659
Krumlauf
R.
(
1994
)
Hox genes in vertebrate development.
Cell
78
,
191
201
Mackem
S.
,
Mahon
K. A.
(
1991
)
Ghox 4. 7: a chick homeobox gene expressed primarily in limb buds with limb-type differences in expression.
Development
112
,
791
806
Marigo
V.
,
Johnson
R. L.
,
Vortkamp
A.
,
Tabin
C. J.
(
1996
)
Sonic hedgehog differentially regulates expression of Gli and Gli3 during limb development.
Dev. Biol
180
,
273
283
Masuya
H.
,
Sagai
T.
,
Moriwaki
K.
,
Shiroishi
T.
(
1997
)
Multigeniccontrol 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
Mo
R.
,
Freer
A. M.
,
Zinyk
D. L.
,
Crackower
M. A.
,
Michaud
J.
,
Heng
H. H.-Q.
,
Chik
K. W.
,
Shi
X.-M.
,
Tsui
L.-C.
,
Cheng
S. H.
,
Joyner
A. L.
,
Hui
C.-C.
(
1997
)
Specific and redundant functions of Gli2 and Gli3 zinc finger genes in skeletal patterning and development.
Development
124
,
113
123
Morgan
B. A.
,
Izpisua-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
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
66
Noramly
S.
,
Pisenti
J.
,
Abbott
U.
,
Morgan
B.
(
1996
)
Gene expression in the limbless mutant- polarized gene expression in the absence of Shh and an AER.
Dev. Biol
179
,
339
346
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
Rooker
G. D.
(
1979
)
The embryological congruity of the human hip joint.
Ann. R. Coll. Surg. Engl
61
,
357
361
Ros
M. A.
,
Lyons
G. E.
,
Mackem
S.
,
Fallon
J. F.
(
1994
)
Recombinant limbs as a model to study homeobox gene regulation during limb development.
Dev. Biol
166
,
59
72
Ros
M. A.
,
Lopez-Martinez
A.
,
Simandl
B. K.
,
Rodriguez
C.
,
Izpisua-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
Schimmang
T.
,
Lemaistre
M.
,
Vortkamp
A.
,
Ruther
U.
(
1992
)
Expression of the zinc finger gene Gli3 is affected in the morphogenetic mouse mutant extra-toes (Xt).
Development
116
,
799
804
Schughart
K.
,
Bieberich
C. J.
,
Eid
R.
,
Ruddle
F. H.
(
1991
)
A regulatory region from the mouse Hox-2. 2 promoter directs gene expression into developing limbs.
Development
112
,
807
811
Small
K. M.
,
Potter
S. S.
(
1993
)
Homeotic transformations and limb defects in Hox A11 mutant mice.
Genes Dev
7
,
2318
2328
van der Hoeven
F.
,
Zakany
J.
,
Duboule
D.
(
1996
)
Gene transpositions in the HoxD complex reveal a hierarchy of regulatory controls.
Cell
85
,
1025
1035
Yokouchi
Y.
,
Nakazato
S.
,
Yamamoto
M.
,
Goto
Y.
,
Kameda
T.
,
Iba
H.
,
Kuroiwa
A.
(
1995
)
Misexpression of Hoxa-13 induces cartilage homeotic transformation and changes cell adhesiveness in chick limb buds.
Genes Dev
9
,
2509
2522
Yokouchi
Y.
,
Sasaki
H.
,
Kuroiwa
A.
(
1991
)
Homeobox gene expression correlated with the bifurcation process of limb cartilage development.
Nature
353
,
443
445
Zakany
J.
,
Duboule
D.
(
1996
)
Synpolydactyly in mice with a targeted deficiency in the HoxD complex.
Nature
384
,
69
71
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