Unité de Génétique Moléculaire de la Morphogenèse, Institut Pasteur, URA 2578 du CNRS, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France
Author for correspondence (e-mail:
brobert{at}pasteur.fr)
Accepted 25 April 2005
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SUMMARY |
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Key words: Mouse, Limb, Anteroposterior patterning, Dorsoventral specification, Bmp signalling, Homologous recombination
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Introduction |
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Proximodistal (PD) development depends on the apical ectodermal ridge
(AER), a transient epithelial structure rimming the distal margin of the limb
bud (Saunders, 1948;
Summerbell, 1974
) that
provides molecular signals to sustain cell proliferation in the underlying
mesenchyme. Fgf8 and Fgf4 (and to a lesser extent
Fgf9 and Fgf17) are expressed in the AER and promote normal
limb development by maintaining the expression of another Fgf family member,
Fgf10, in the mesenchyme (Sun et
al., 2002
). Reciprocally, in Fgf10-/- mutant
embryos, the AER does not form, leading to a complete truncation of fore- and
hindlimbs (Sekine et al.,
1999
). Thus, it appears that PD limb outgrowth is dependent upon a
positive feedback between the AER- and apical mesoderm-specific Fgf
activities.
The key organizer for the anteroposterior (AP) axis has been identified as
a population of cells from the posterior mesenchyme, called the zone of
polarizing activity (ZPA), that specifically secretes the sonic hedgehog (Shh)
protein (reviewed by Pearse and Tabin,
1998). Heterotopic grafting of the ZPA
(Saunders and Gasseling, 1968
)
or of Shh-expressing cells
(Riddle et al., 1993
) to the
anterior margin of the limb bud leads to a mirror duplication of the digits.
Conversely, the Shh null mutation provokes a dramatic loss of the
anterior skeleton of the autopod (handplate/footplate) where a single digit I
remains (Chiang et al., 2001
).
However, although Shh is necessary for normal expansion of the AP axis, the
limb field is prepatterned along the AP axis prior to Shh activation
through mutual genetic antagonism between Gli3 and Hand2
(te Welscher et al.,
2002
).
Dorsoventral (DV) asymmetry is regulated, after limb bud formation, by the
ectoderm. The dorsal and ventral limb ectoderm domains express, respectively,
Wnt7a and En1. Mutations in both these genes have revealed
that En1 is necessary to repress Wnt7a expression in the
ventral ectoderm and that Wnt7a is the key inducer of Lmx1b in the
dorsal mesenchyme. Thus, the En1 mutation leads to the ectopic
ventral expression of Wnt7a in the ectoderm and Lmx1b in the
mesoderm, and the distal structures develop with bi-dorsal characteristics
(Loomis et al., 1996;
Cygan et al., 1997
).
Conversely, in the absence of Wnt7a, the dorsal pattern of the autopod is not
established and the limb appears biventral
(Parr and McMahon, 1995
).
These three organizing centres do not work independently. Rather,
patterning and growth of the limb in three dimensions is coordinated through
complex interactions between the different limb organizing centres (reviewed
by Niswander, 2002). For
example, AER induction depends not only on lateral plate-produced Fgfs but
also on DV ectoderm polarity. This is related to Bmp signalling which is
required both for DV patterning and AER induction
(Ahn et al., 2001
;
Pizette et al., 2001
).
Msx genes encode homeodomain transcription factors and their expression is
associated with epithelio-mesenchymal interactions at many sites in vertebrate
embryos such as limb buds, craniofacial processes and tooth buds
(Hill et al., 1989;
Robert et al., 1989
) (reviewed
by Davidson, 1995
). Knockout
experiments have shown that Msx1-null mutations provoke defects in
craniofacial development, cleft palate, inner ear malformations and tooth
agenesis (Satokata and Maas,
1994
; Houzelstein et al.,
1997
), whereas Msx2 null mutations lead to abnormal tooth
development, loss of fur and reduced and disorganized cerebellar lobules
(Satokata et al., 2000
) (Y.L.,
M-A.N., A.B. and B.R., unpublished). Single Msx homozygous mutants
(Msx1-/- or Msx2-/-) do not display
gross limb abnormalities. However, several lines of evidence suggest that this
gene family may play a crucial role in limb morphogenesis. First, both genes
are prominently expressed in the limb field from the earliest stages of limb
formation and, later on, in both the AER and the subjacent mesenchyme.
Furthermore, they require functional interactions between ectoderm and
mesoderm for their expression and have been proposed to mediate transduction
of inductive signals at this site
(Davidson et al., 1991
;
Robert et al., 1991
). Second,
misexpression of Msx1 in dorsal ectoderm of the chick limb bud can
induce formation of ectopic AERs, similarly to misexpression of a
constitutively activated Bmp receptor, suggesting that Msx1 acts downstream of
Bmp signals in AER induction (Pizette et
al., 2001
).
The co-expression of Msx1 and Msx2 and their possible overlapping functions might explain the absence of a limb phenotype in either of the simple mutants. We have analysed Msx1-/-; Msx2-/- double mutant embryos. We observed that Msx genes play a role at several stages of limb development and that their mutation affects the three organizing centres. In the double mutant, dorsoventral patterning is disturbed at the anterior border of the limb bud and this precludes AER formation anteriorly. Msx genes are necessary for the regression of the AER at later stages. The mutation of the two genes leads to further malformations along the AP axis, initially by preventing the development of the anteriormost region of the autopod (distal segment) and zeugopod (intermediate segment) and, later on, by provoking the abnormal overgrowth of the remaining anterior part of the autopod mesenchyme. These defects can be explained in the context of a role for Msx genes in Bmp signal transduction.
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Materials and methods |
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In situ hybridization
Whole-mount in situ hybridization was performed as described previously
(Houzelstein et al., 1997).
RNA probes were generated from the following DNA fragments: Fgf8 was
a complete cDNA sequence obtained by PCR (forward primer CGC TCG GGC TCT CAG
TGC TCC; reverse primer: GAG CTG GGC GAG CGC CTA TCG). Alx4 was a
gift from F. Meijling; Hoxd11 from P. Dollé; Fgf4
from G. Martin; Fgf9 from R. Kelly; Shh from A. McMahon;
En1 from K. Schughart; Lmx1b from R. Johnson; Bmp2,
Bmp4 and Bmp7 from B. Hogan; gremlin from R. Zeller; and
Pax9 from R. Balling.
Histology, cell death analysis and skeletal preparation
Skeletal preparation was as described in Zhang et al.
(Zhang et al., 1995).
Histological sections were carried out as described in Kaufman
(Kaufman, 1994
). For
whole-mount cell death detection, embryos were fixed overnight in 4%
paraformaldehyde and then dehydrated in methanol for long-term storage at
-20°C. TUNEL analysis was performed on rehydrated embryos using the
ApopTag Peroxidase detection kit (Q.BIOgene) according to the manufacturer's
protocol for tissue cryosections and cells.
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Results |
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Abnormalities along the anteroposterior axis were also very severe. They
were confined to the anterior elements of the skeleton and displayed
apparently paradoxical features. The main feature was a general truncation of
the anterior part of the limbs with an almost systematic loss of the anterior
element of the zeugopod and a frequent oligodactyly
(Fig. 1A,B)
(Table 1). In oligodactylous
animals, the missing digit was always the anteriormost one (digit I). Carpals
and tarsals were also affected in the anterior region. The posterior elements
were present, the medium elements severely reduced or barely visible, and the
anteriormost ones absent (Fig.
1C,D). Connections were maintained with the remaining digits,
which allowed confirmation of their identity. In addition, double-null embryos
lacked the pubis bone (data not shown), which corresponds to the anterior part
of the pelvis, based on phylogenetic and embryological considerations
(Hinchliffe and Johnson, 1980;
Knezevic et al., 1997
).
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AER maturation but not initiation is affected in double-null mutants
The size of double-null limb buds was reduced by about one third compared
with controls at 10.5 to 11.5 dpc, even in somite-matched embryos (compare,
for example, Fig. 3E with
3F, Fig.
3G with
3H or Fig.
4G with
4H). This prompted us to
investigate the activity of the AER. Fgf8 is expressed specifically
in pre-AER and AER cells (Loomis et al.,
1998). Fgf8 expression was detected as early as the
14-somite stage in the double-null embryo limb field, indicating that AER
initiation took place at the normal stage
(Fig. 2A,B). However, its
subsequent maturation was impaired. During mouse limb development, the cells
giving rise to the AER are recruited from the ventral part of the limb bud
ectoderm to form a broad pre-AER domain that becomes progressively thinner and
ends as a thin strip of cells at the tip of the bud
(Loomis et al., 1998
). In
double-null embryos, at 10.5 and 11.5 dpc, the AER appeared shorter and more
diffuse than normal (Fig. 2C-H) as shown by Fgf8 (Fig.
2C,D) or Bmp7 (Fig.
2E,F) expression profiles, and histological analysis
(Fig. 2 G,H). In addition,
Fgf4 (Fig. 2I,J) and
Fgf9 (data not shown), two other Fgf genes normally expressed in the
AER as early as 10.5 dpc, were barely detectable at this stage in double-null
embryos, proving that the activity of the AER was also delayed.
The anteriormost part of the double-null mutant limb regresses before the twelfth day of development
At 11.5 dpc, a sharp discontinuity was visible at the anterior of the limbs
that appeared truncated anteriorly (Fig.
3A,B; see also Fig.
7C,D). At this stage, Fgf8 is normally expressed over the
entire AER, while Fgf4 expression is absent from its anterior third
(Niswander and Martin, 1992;
Crossley and Martin, 1995
)
(see also Fig. 6A). These
markers were analysed together in double in situ hybridisation. At 11.5 dpc,
Fgf4 was expressed at a normal level in double-null limb buds but its
expression pattern was abnormal relative to that of Fgf8. As
expected, the AER of control embryos displayed an anterior domain expressing
Fgf8 (Fig. 3A, red
staining) but devoid of Fgf4-specific signal
(Fig. 3A, purple staining). In
double-null mutant embryos, Fgf8 and Fgf4 expression domains
abutted exactly to the same anterior limit in the AER, which corresponded to
the anterior limit of the ridge and to the sharp angular anterior border of
the misshaped limb buds (Fig.
3B). These observations suggested that the anterior third of the
AER was missing at this stage. In addition, the Fgf4 expression
domain was shifted anteriorly (Fig.
3B; see also Fig.
6B).
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These results, together with the morphological aspect of the double-null limb buds, demonstrate that the region corresponding to the anteriormost digit disappears between 10.5 and 11.5 dpc.
The dorsoventral polarity of the limb bud is altered anteriorly in double-null mutant embryos, precluding AER formation in this region
To analyse the possible involvement of Msx genes in DV polarity, we
resorted to two markers of limb DV asymmetry. The first one, En1, is
specifically expressed in the ventral ectoderm and the ventral AER of the limb
bud, as early as 9.5 dpc (Loomis et al.,
1998). At 9.75 dpc (24 somites)
(Fig. 4A,B) and 10.5 dpc (data
not shown), En1 expression was reduced in double-null limbs. However,
at 11.5 dpc, En1 signal was detected similarly in control and
double-null embryos, with the exception of the anterior part of the AER, which
is missing at this stage in the double mutant
(Fig. 4C,D). The second one,
Lmx1b, is a specific marker of the dorsal mesenchyme and a read-out
of ectodermal Wnt7a activity (Riddle et
al., 1995
; Cygan et al.,
1997
). At 9.5 dpc, no obvious abnorma1ity in Lmx1b
expression could be detected (data not shown) but, as early as 10.5 dpc, an
ectopic ventral expression domain was visible at the anterior aspect of the
double-null limb bud (Fig.
4E,F). This result was confirmed at 11.5 dpc. At this stage, the
anterior ectopic expression domain corresponded precisely to the part of the
limb that is devoid of AER (Fig.
4G-I). Thus, anteriorly, the normal DV boundary is not established
in the double-null embryo limb bud. Considering the relationship between DV
boundary formation and AER induction
(Zeller and Duboule, 1997
;
Pizette et al., 2001
), this is
the most likely explanation for the absence of AER anteriorly. As the AER is
required to maintain the underlying mesenchyme
(Dudley et al., 2002
), this
would lead, at later stages, to the loss of anterior mesenchyme and truncation
of the anterior skeleton elements.
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A later effect on anteroposterior polarity in double-null mutant limbs
In addition to AER persistence, overgrowth of the anterior-distal
mesenchyme of the handplate was observed from 11.75 dpc in a large proportion
of double-null mutant limbs, which may underlie the polydactyly observed at
later stages (compare, for example, Fig.
5G with
5H, Fig.
6C with
6D, Fig.
6G with
6H or Fig.
7G with
7H). This was mainly observed
in the forelimb whereas the hindlimb generally displayed only a discrete
anterior deformation (compare Fig
5E with
5F or Fig.
6E with
6F). In addition, the extent of
the outgrowth was variable from one individual to another and sometimes from
one side to the other in the same embryo.
The first sign of this abnormal limb polarisation was indicated by
Fgf4 expression. In the normal mouse at 11.5 dpc, Fgf4
expression is observed in the apicoposterior part of the AER, but excluded
from the ridge overlying the Shh- expressing mesenchymal domain
(Lewandoski et al., 2000). In
the double-null mutant, the Fgf4 domain was slightly shifted
anteriorly, correlatively with the loss of the anterior part of the AER and
the anterior extension of the Shh expression domain
(Fig. 6A,B,I,J). By 11.75 dpc,
the remaining part of the AER of the double-null mutant appeared to extend
anteriorly (data not shown). The expression of Fgf4, which normally
disappears at this stage, persisted anteriorly in the double-null mutant AER
and was still detectable at its anterior tip up to 12.5 dpc, whereas in
control embryos, Fgf4 expression had been switched off for more than
12 hours. This persistent expression domain corresponded to the tip of the
anterior deformation observed at this stage
(Fig. 6C,D).
Correlatively, overgrowth of the mesenchyme was observed, mainly in forelimbs. As the anterior outgrowth appeared secondarily on truncated limb buds devoid of anterior mesenchyme, we investigated the identity of outgrowing cells using the Hoxd11 marker. At 12.5 dpc, similar to 11.5 dpc, a prominent expression was observed in the anterior outgrowth of the limb in double-null embryos (Fig. 6F,H). Therefore, the whole limb bud mesenchyme of the double mutants assumes a posterior identity. Our interpretation is that the anterior region, being devoid of AER, does not grow out and even regresses. Secondarily, persistent Fgf4 activity at the anterior end of the AER together with Fgf8 activity may lead to the overgrowth of the remaining anterior mesenchyme, resulting in the anomalous shape of the limb observed from 12.5 dpc onwards. Proliferation rate was not noticeably enhanced, as judged by expression of phosphorylated histone H3 or cyclin D1 (data not shown). However, at this stage, all limb bud cells are actively dividing, and a slight increase in the proliferation rate anteriorly would not be detected by these methods.
In the limb, posterior identity is associated with the expression of
Shh, such that its anterior misexpression leads to pre-axial
polydactyly (reviewed by Hill et al.,
2003). In the double-null, Shh was weakly expressed at
10.5 dpc (data not shown) but its expression level was similar to control at
11.5 dpc (Fig. 6I,J). At this
stage, in accordance with the normal expression of other mesenchymal markers
posteriorly, the domain of Shh expression was normal in most cases,
with the exception of a slight anterior extension
(Fig. 6I,J). However, in some
embryos, a small anterior ectopic domain of Shh expression could be
observed, exclusively in the forelimbs (four forelimbs out of 14 recovered at
11.5 dpc) (Fig. 6J). The extent
of the anterior outgrowth at 12.5 dpc may correlate with the intensity of
Shh ectopic expression at earlier stages, but no direct evidence can
be provided at present to support this hypothesis. In hindlimbs, Shh
expression appeared as a single posterior domain but slightly displaced
distally (not shown).
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At 12.5 dpc, Bmp4 expression, in the mesenchyme, is mainly concentrated in the interdigital spaces and digit anlagen. In the mutant, the Bmp4 transcript level was not altered, with the exception of a slight decrease in the interdigital webbing (data not shown). At 13.5 dpc, Bmp4 expression was present in the digit anlagen of both controls and mutants (Fig. 7E,F). It was also observed in the distal mesenchyme of control embryos (Fig. 7E, arrowhead), but not in the mutant (Fig. 7F), suggesting that Msx genes act upstream of Bmp4 at this site. In addition, expression was observed in the AER that remained in the double-null mutant (Fig. 7F).
Bmp2, contrary to Bmp4, is not normally expressed in the anterior part of the autopod. Similarly, Bmp7 is not expressed anteriorly from 13.5 dpc. In the double-null mutant, at 13.5 dpc, the two genes were also expressed in the anterior domain, in keeping with its posterior identity. Outside this domain, expression pattern was similar but more intense in the mutant when compared with control embryos (Fig. 7G-J).
Gremlin expression was not significantly modified in the double mutant, except that it persisted somewhat longer in the interdigital spaces. Thus, gremlin expression was undetectable at 13.5 dpc in both the fore and hindlimbs of control embryos (Fig. 7K and data not shown) but was still detectable, at a low level, in the interdigital spaces of the sole hindlimbs of the mutants (Fig. 7L and data not shown).
In conclusion, Bmp expression was not significantly modified by the Msx gene mutation. In the mutant, phosphorylation of the Smad1, 5 and 8 proteins could be detected at 10.5, 12.5 and 13.5 dpc, wherever one of the Bmp genes is expressed (data not shown), indicating that Msx proteins do not interfere with the first steps of Bmp signalling.
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Discussion |
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In the limb, a number of data have shown that Msx genes are downstream
targets of Bmp signalling. At early stages, Msx1 and Msx2
are expressed in nearly identical patterns that overlap significantly with Bmp
genes, namely Bmp2, Bmp4 and Bmp7
(Ahn et al., 2001;
Pizette et al., 2001
).
Enhanced Bmp signalling, which is observed in animals heterozygous for the
Fused toes (Ft) mutation (Heymer
and Rüther, 1999
), or in mutants homozygous for a null allele
of gremlin, a potent Bmp antagonist
(Khokha et al., 2003
), leads
to an upregulation of both Msx1 and Msx2 expression.
Reciprocally, blocking Bmp signalling in the limb ectoderm provokes a decrease
of Msx2 expression (Wang et al.,
2004
). Our data show that, during all phases of limb development,
the absence of Msx activity leads to phenotypes that in many respects mimic
the absence or decrease in Bmp signalling: delay in both maturation and
regression of the AER (Pizette and
Niswander, 1999
; Wang et al.,
2004
); anterior upregulation of Fgf4 expression in the
AER (Zùniga et al.,
1999
); tendency to anterior polydactyly
(Hofmann et al., 1996
;
Dunn et al., 1997
;
Katagiri et al., 1998
); and
absence of interdigital tissue regression
(Guha et al., 2002
;
Wang et al., 2004
). The
absence of DV specification anteriorly and the loss of the corresponding part
of the AER reproduce locally the effect of Bmp-receptor inhibition
(Ahn et al., 2001
;
Pizette et al., 2001
). In limb
development, Msx genes seem to act downstream of Bmp signalling, since Bmp
expression profiles are little affected in the double mutant, as is the case
for Smads.
AER maturation but not initiation is affected in Msx1-/-; Msx2-/- double mutants
The AER is established at the boundary between dorsal and ventral ectoderm
(Altabef et al., 1997;
Michaud et al., 1997
;
Tanaka et al., 1998
;
Kimmel et al., 2000
). At the
molecular level, this boundary takes place between Bmp-expressing
(ventrally) and non-expressing (dorsally) domains at the stage when limb buds
form. In the chick, Pizette et al.
(Pizette et al., 2001
) have
demonstrated that depletion of the ventral Bmp signal prevents En1
expression in the ventral ectoderm and precludes AER formation. Reciprocally,
activation of Bmp signalling over the whole limb ectoderm leads to induction
of En1 also in the dorsal ectoderm, which adopts a ventral identity,
and abrogates AER formation. In addition, activated Bmp receptor gene
expression in the dorsal ectoderm induces patches of Fgf8 expression.
Based on these and other results, these authors have proposed that Bmp
signalling is governing both DV limb patterning and AER formation via the
induction of En1 and Msx genes, respectively, and that these two
pathways are independent. Furthermore, they proposed that Msx genes are
downstream effectors of the Bmp signal in AER formation. This is supported by
two main lines of evidence: (1) Msx1 and Msx2 display
overlapping expression patterns with Bmp4 and Bmp7 in
ventral ectoderm at early stages of limb development; and (2) ectopic
expression of Msx1 in limb dorsal ectoderm, where Msx genes are
normally not expressed at stages of AER induction, may result in the formation
of ectopic ridges expressing Fgf8. These extra ridges would be
induced by the formation of a new boundary between Msx-expressing and
non-expressing cells. In accordance with this model, in the mouse, conditional
inactivation of the Bmpr1a gene in limb ectoderm also elicits both DV
patterning defects, involving En1 downregulation, and the absence of
AER formation (Ahn et al.,
2001
).
Our data confirm that Msx play an essential role in AER formation.
Nevertheless, this role does not fit exactly with the model proposed by
Pizette et al. (Pizette et al.,
2001). First, in the absence of Msx proteins, AER is induced and
maintained over the major part of the limb bud apex, implying that Msx
function is required for AER formation only at the anterior margin. Second, in
the anterior region where the AER does not form, this is correlated with the
absence of a DV boundary, as, based on the expression patterns of
Lmx1b, the bud is bi-dorsal in this domain. Therefore, the deficiency
in AER formation does not appear to be independent of DV patterning.
In the more posterior part of the double-null limb, the AER forms, but its
maturation is delayed and it remains wider than normal. This too may result
from impairment in Bmp signalling. Misexpression of noggin from a transgene in
the AER and over the limb ventral ectoderm, in the mouse, also leads to
ventral extension of the AER (Wang et al.,
2004). Our results suggest that in the absence of Msx proteins,
Bmp signalling is affected along the whole apex of the limb. Anteriorly, this
would result in loss of the AER, agenesis of mesoderm and lack of skeletal
elements, but posteriorly only to incomplete AER maturation. Posteriorly, a
deficit in Bmp signalling may be of limited effect or, conversely, other genes
may fulfil the role of Msx.
During digit individualization, regression of the AER and of interdigital tissues is impaired
From 12.5 dpc to 14.5 dpc, the double-null limb phenotype is characterized
by the slow and incomplete regression of the AER and the persistence of the
interdigital soft tissues, preventing correct individualization of the digits.
Regression of the AER is under the control of Bmp signalling. In the chick,
retroviral expression of the Bmp antagonist noggin inhibits AER regression.
Accordingly, from stage 24, Msx2 is downregulated in the AER and
underlying mesenchyme, whereas Msx1 is downregulated in the posterior
part of the apical mesenchyme (Pizette and
Niswander, 1999). In the mouse, expression of noggin, from a
transgene, in the ectoderm leads to both persistence of the AER and
downregulation of Msx2 in this structure but not in the mesenchyme,
whereas Bmp4 expression is not modified
(Guha et al., 2002
;
Wang et al., 2004
). Thus,
during AER regression, Msx genes are probably under the control of Bmp
signalling. Our data further suggest that they are required to transduce Bmp
activity in the AER because, in the Msx1-/-;
Msx2-/- double-null mutants, AER regression is impaired from
12.5 dpc while Bmp4 expression is not modified along the apical
border of the limb at this stage.
The involvement of Msx genes in interdigital tissue regression by apoptosis
has been proposed by several authors (reviewed by
Chen and Zhao, 1998). Both
Msx1 and Msx2 are expressed in the interdigital tissue at
the time when apoptosis takes place
(Robert et al., 1989
;
Coelho et al., 1991b
).
Furthermore, this expression pattern correlates with the extent of
interdigital tissue regression in different species
(Gañan et al., 1998
).
Our results confirm this role because, in the double-null mutant, interdigital
tissue webbing fails to regress. Bmp proteins have been identified as the
trigger in this process (Yokouchi et al.,
1996
; Zou and Niswander,
1996
; Macias et al.,
1997
; Guha et al.,
2002
). Whether Msx gene expression is required upstream or
downstream of Bmp signalling is controversial
(Ferrari et al., 1998
;
Merino et al., 1999
). Our
results suggest that, in interdigital webbing regression, Msx would act
downstream of Bmp signalling as expression of Bmp2 and Bmp7 is unchanged and
that of Bmp4 only slightly decreased at the stage when apoptosis is
active.
Roles of Msx genes in regulating the anteroposterior polarity of the limb
Between 11.5 and 12.5 dpc, the double-null handplate, particularly in
forelimbs, starts undergoing an important anterior overgrowth. This is
correlated with a prominent anterior extension of the AER and persistence of
Fgf4 and Fgf8 expression up to the anteriormost limit of the
elongated AER. Experimental evidence shows that whenever the AER is elongated,
or Fgf4 signalling increased, by genetic manipulation, this leads to an
overgrowth of the underlying mesenchyme and later on, to anterior polydactyly
(e.g. Hofmann et al., 1996;
Liu et al., 2003
;
Wang et al., 2004
). This is
probably due to the mitogenic properties of Fgf proteins on limb mesenchyme
(reviewed by Martin, 1998
) but
also to the capacity of Fgf4 to recruit cells to the limb bud from adjacent
territories (Tanaka et al.,
2000
). Noticeably, the digits duplicated anteriorly display, in
some cases, a posterior identity (Hofmann
et al., 1996
; Liu et al.,
2003
).
Anterior outgrowth, leading to polydactyly, may also be due to impairment
in the transduction of Bmp signal in the absence of Msx function. Anterior
polydactyly is often observed in situations where Bmp signalling is globally
diminished, such as in heterozygous Bmp4-null mutants
(Dunn et al., 1997), in
Bmp7 homozygous mutants (Luo et
al., 1995
; Hofmann et al.,
1996
) or in Bmp4+/-;
Bmp7+/- double heterozygous mutants
(Katagiri et al., 1998
). This
is not necessarily associated with ectopic expression of Shh
anteriorly (Dunn et al.,
1997
). Nevertheless, Bmp4 has been shown to repress Shh
in the tooth epithelium and to act downstream of Msx1 in this
process. This property is conserved in the limb, although a role for
Msx1 at this site was not investigated
(Zhang et al., 2000
). We
observed only a discrete and inconsistent ectopic expression of Shh
at the anterior of mutant limb buds. However, this was observed only in the
forelimb bud, which tends to produce more frequent and more extensive
polydactylies than the hindlimb. In addition, the proportion of limb buds with
an ectopic Shh domain corresponds approximately to that of
polydactylous limbs at later stages. Furthermore, Shh upregulation
may be undetectable by in situ hybridization, but nonetheless sufficiently
high to induce extra digits, as exemplified by the raz mutant
(Krebs et al., 2003
).
Therefore, it remains possible that Msx genes are required to repress
Shh anteriorly, either directly or by mediating the Bmp signal.
Conversely, Msx genes may play a crucial role in the mechanisms that
pattern the anterior limb region. Recent data have shown that Shh is
instrumental in patterning digits V to II, and ulna/fibula in the zeugopod
(reviewed by Zeller, 2004).
The determinants of more anterior structures remain to be identified, and
these are precisely the structures affected in the Msx1; Msx2 double
null mutant.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/132/13/3003/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Depto de Biologia Celular, Facultad de Biologia, Av.
Diagonal, 645, 08071 Barcelona, Spain
Present address: INSERM U679, Neurobiologie et Thérapeutique
Expérimentale, Hôpital de la Salpêtrière, 47
boulevard de l'Hôpital, 75013 Paris, France
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