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First published online 22 February 2006
doi: 10.1242/dev.02298
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1 Research Institute of Molecular Pathology (IMP), Dr Bohr-Gasse 7, A-1030
Vienna, Austria.
2 Department of Pharmacology, Graduate School of Medicine, Kyoto University,
Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.
3 Max Delbrück Center for Molecular Medicine, Robert-Roessle-Strasse 10,
13125 Berlin, Germany.
* Author for correspondence (e-mail: hartmann{at}imp.univie.ac.at)
Accepted 24 January 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: ß-Catenin, Limb patterning, Scapula, Emx2, AER, Lmx1b, BMP, Dorsoventral axis
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Members of the
Wingless/Int (Wnt) family of secreted signaling molecules have been shown to
play important roles in both of these processes (Church et al., 2002;
Yang, 2003). In chick,
Wnt2b and Wnt8c have been implicated in Fgf10
activation via the canonical Wnt/ß-catenin pathway, thereby inducing limb
bud outgrowth (Kawakami et al.,
2001; Ng et al.,
2002). Fgf10 in the mesenchyme is required for induction
of the apical ectodermal ridge (AER) marker Fgf8 in a strip of flank
ectoderm before the formation of a morphological AER
(Martin, 1998). The AER is a
strip of stratified epithelia at the distal limb bud margin that is essential
for limb outgrowth and for proximodistal patterning
(Saunders, 1948). Induction of
Fgf8 by mesenchymal Fgf10 is thought to be mediated by
ectodermal Wnt signals, such as Wnt3a in chick and Wnt3 in
mouse, which activate and maintain the expression of Fgf8 in the AER
through the canonical Wnt/ß-catenin pathway
(Barrow et al., 2003;
Kengaku et al., 1998;
Soshnikova et al., 2003). At
least two of the ß-catenin co-factor genes, lymphoid enhancer binding
factor 1 (Lef1) and T-cell factor 1 (Tcf1; Tcf7 -
Mouse Genome Informatics), are required for Fgf8 expression in mice
(Galceran et al., 1999).
However, in Lef1/Tcf1 double-mutant mice, Fgf10 expression
is initiated normally, which might be due to residual canonical Wnt signaling,
as two other members of the Tcf family, Tcf3 and Tcf4
(Tcf7l2 - Mouse Genome Informatics), are still present
(Agarwal et al., 2003). Signals
from the AER are involved in two important feedback loops, one with the distal
mesenchyme required for AER maintenance and further outgrowth of the limb; the
other one with the zone of polarizing activity (ZPA), which is important for
AER regression (Niswander,
2003; Tickle,
2003). In the latter, Fgf4, probably together with other
Fgfs, maintains the expression of Shh in the ZPA
(Chiang et al., 1996;
Laufer et al., 1994).
Shh in turn downregulates Bmp activity by directly or indirectly
inducing the Bmp-antagonist gremlin (Grem1 - Mouse Genome
Informatics) (Khokha et al.,
2003). Without gremlin, the AER regresses prematurely as a result
of the downregulation of Fgf4 (and probably Fgf9 and
Fgf17) by Bmps in the posterior AER
(Guha et al., 2002;
Khokha et al., 2003;
Pizette and Niswander, 1999;
Zuniga et al., 1999;
Zuniga et al., 2004). During
normal limb development, this feedback loop stops, leading to regression of
the AER when regions of the limb mesenchyme can no longer respond to
Shh and, thus, gremlin becomes downregulated
(Scherz et al., 2004).
In addition, Wnt signaling is required for dorsoventral patterning of the
limb mesenchyme. Wnt7a is expressed only in the dorsal ectoderm
because of the activity of the ventral ectodermally expressed engrailed 1
(En1) (Loomis et al.,
1996; Parr and McMahon,
1995). Wnt7a regulates expression of the transcription
factor Lmx1b, known as Lmx1 in chick, presumably through a
non-canonical signaling pathway (Cygan et
al., 1997; Kengaku et al.,
1998; Riddle et al.,
1995; Vogel et al.,
1995). Lmx1b, expressed exclusively in the dorsal
mesenchyme, is required for the specification of dorsal limb identity in mouse
and chick (Chen et al., 1998;
Dreyer et al., 1998;
Riddle et al., 1995;
Vogel et al., 1995).
Previous loss-of-function studies in mouse addressing the role of canonical
Wnt-signaling in limb patterning either solely focused on its role in the
ectoderm, or did not allow the differences between the mesenchymal and the
ectodermal contribution to be distinguished
(Barrow et al., 2003;
Galceran et al., 1999;
Soshnikova et al., 2003).
Therefore, we specifically addressed the contribution of mesenchymal
ß-catenin during early limb development using conditional loss- and
gain-of-function alleles and the limb mesenchyme-specific Prx1-Cre line. Here,
we show that canonical Wnt signaling in the mesenchyme is required for
maintaining Fgf10 and Fgf8 expression, which suggests a
specific role for mesenchymal ß-catenin in AER maintenance, in addition
to the one proposed in the ectoderm (Barrow
et al., 2003; Soshnikova et
al., 2003). Interestingly, we also identified a novel role for
active Wnt signaling in AER regression. This effect is concomitant with an
upregulation of Bmps and Bmp target gene expression, suggesting that canonical
Wnt signaling lies upstream of Bmps in the limb mesenchyme.
Unexpectedly, we found that mesenchymal ß-catenin is involved in regulating the dorsal determinant Lmx1b. Loss of ß-catenin activity results in a reduced and partial loss of Lmx1b expression, whereas expression of a dominant-active form results in its expansion; however, Wnt7a and En1 remain correctly localized in the dorsal and ventral ectoderm, respectively.
Finally, we found that, unlike many other mutants that affect limb bud
outgrowth, loss of mesenchymal ß-catenin results in agenesis of the
scapula, despite the presence of a humeral-like element. This phenotype is
probably primarily due to the loss of expression of the homeobox gene
Emx2 in ß-catenin mutant limbs, as Emx2 mutants also
display severe scapula agenesis
(Pellegrini et al., 2001).
Emx2 is upregulated in the gain-of-function limbs and is therefore
likely to be under the direct control of canonical Wnt signaling.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), to
females homozygous for a floxed allele (fl)
(Huelsken et al., 2001), to
generate Prx1Cre/+;ß-catfl/- embryos
[hereafter called ß-cat
Prx1/- or loss of
function (lof)]. Gain-of-function embryos were generated by mating female mice
homozygous for the ß-catenin exon3 floxed allele (ex3fl)
(Harada et al., 1999) to
Prx1Cre heterozygous males to produce
Prx1Cre/+;ß-catex3fl/+ embryos [hereafter
called ß-catex3Prx1/+ or gain-of-function (gof)].
Prx1-Cre line activity was analyzed by crossing males to Z/AP
reporter females (Lobe et al.,
1999). Mouse embryos were collected and staged either by counting
somites or according to Theiler (Theiler,
1989). Genotypes of the embryos were determined by PCR and by
morphology. Embryos were fixed in 4% paraformaldehyde/PBS at 4°C
overnight, washed, and dehydrated into either 100% methanol for whole-mount in
situ hybridization or 100% ethanol for paraffin wax embedding. Paraffin
sections were cut at 5 µm. Whole-mount alkaline phosphatase staining was
performed as described previously (Lobe et
al., 1999).
Skeletal preparations and scanning electron microscopy (SEM)
Embryos were skinned, eviscerated and fixed in 95% ethanol. Skeletons were
stained with Alizarin Red and Alcian Blue, or just with Alcian Blue
(McLeod, 1980). For SEM
analysis embryos were fixed overnight in 2% glutaraldehyde/0.1 M sodium
cacodylate buffer (SCB), pH 7.2, washed in SCB, postfixed for 90 minutes in 1%
osmium tetroxide, washed in SCB, and dehydrated into 100% ethanol and
critical-point dried.
Whole-mount, section and double-fluorescent in situ hybridisation
Whole-mount and section in situ hybridisation was performed as described
previously (Murtaugh et al.,
1999; Riddle et al.,
1993). For sectioning of whole-mount, stained embryos, embryos
were embedded in gelatin and vibratome sectioned at 20 µm.
Double-fluorescent in situ hybridisations on paraffin sections were performed
using biotin- and DIG-labelled probes. After hybridisation, slides were
washed, quenched and blocked. Probes were detected by incubation with
streptavidine-HRP (Perkin Elmer, diluted 1 in 100) and anti-DIG-HRP (Roche,
diluted 1 in 50), followed by Cy3- or Cy5-tyramide-labelled fluorescent dyes
(according to instructions of the TSA Plus Fluorescent Systems kit, Perkin
Elmer). All probes have been described previously. The minimum number of
specimens analyzed for each marker was three for all stages, except for AER
measurements (see legend to Fig.
3), and two for SEM, unless otherwise stated. All comparative
images are at the same magnification, unless otherwise noted. AER measurements
were performed on Fgf8-stained limbs by measuring the pixel number
for each limb using Metamorph.
Immunohistochemistry and TUNEL assay
Immunohistochemistry was performed on paraffin sections using monoclonal
mouse anti-ß-catenin (BD Transduction Laboratories, diluted 1 in 200)
after heat-induced antigene retrieval. The signal was detected using a
biotinylated secondary antibody (diluted 1 in 250; Vector Laboratories) in
combination with the ABC Kit (Vector Laboratories) and DAB substrate (Sigma).
TUNEL assays were performed using the Flourescein In Situ Cell Death Kit
(Roche).
Micromass cultures
Micromass cultures were performed as described previously
(Hill et al., 2005). Primer
sequences for RT-PCR are available upon request. Total RNA (1 µg), isolated
using TRIZOL from cultures 12 hours and 14 hours after plating and infection,
was used for reverse transcription. Semi-quantitative RT-PCR was performed
using fivefold dilutions of the cDNA template, starting at a 1 in 10
dilution.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Loss-of-function ß-catPrx1/- embryos
(Fig. 1B) died shortly after
birth and had severely shortened fore- and hindlimbs, with respect to wild
type (Fig. 1A). Notably,
hindlimbs developed further than forelimbs, as can be seen in skeletal
preparations at E13.5 and E14.5 (see Fig. S1 in the supplementary material).
Gain-of-function ß-catex3Prx1/+ embryos also
had shortened limbs (Fig. 1C).
Similar to ß-catPrx1/- embryos, forelimbs were
more severely affected than hindlimbs (Fig.
1C). From E13.5 onwards, formation of haematomes in distal regions
could be observed, primarily but not exclusively, in the hindlimbs of both
mutants.
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; Soshnikova et al.,
2003). Therefore, we confirmed, by in situ hybridization using an
antisense probe specific to the floxed exons
(mß-catenin del), that ß-catenin was still
expressed in the ectoderm and the AER in
ß-catPrx1/- limbs
(Fig. 1E-G). Although wild-type
limbs displayed uniform ß-catenin expression at E10.5
(Fig. 1D),
ß-catPrx1/- forelimbs showed a high degree of
deletion in the limb and flank mesenchyme at E10.5
(Fig. 1E, red and black arrow,
respectively), and almost complete deletion at E11.5
(Fig. 1G). By contrast, only
patchy deletion was detected in the hindlimb mesenchyme at E10.5
(Fig. 1F, red arrow). As has
been previously published, by E11.5 deletion had occurred in most mesenchymal
cells of the hindlimb (Hill et al.,
2005). ZAP-reporter analysis of the Prx1-Cre line revealed that
deletion in the forelimb occurred in a mosaic pattern as early as somite stage
15 (see Fig. S2B,B' in the supplementary material). The different
kinetics of deletion observed between fore- and hindlimb
(Fig. 1E-G) (see also
Logan et al., 2002), are
probably responsible for the differences in fore- and hindlimb development of
the mutants.
AER defects upon loss of mesenchymal ß-catenin activity
Loss of ß-catenin in the mesenchyme resulted in severely shortened
fore- and hindlimbs. Scanning electron microscopy (SEM) analyses of the
forelimbs revealed that the ß-catPrx1/- limb
bud was slightly smaller along the anteroposterior (AP) axis at E9.5
(Fig. 2A,A'). At E10.5,
ß-catPrx1/- limbs were growth retarded and
showed notches in the AER (Fig.
2B,B'). Although we did not observe any gross morphological
difference at E9.5, it is possible that molecular changes contributing to the
later phenotype have occurred. We therefore analyzed the expression of genes
involved in limb outgrowth. Fgf8, one of the earliest known
ectodermal markers for limb outgrowth, was expressed in its normal stripe-like
pattern at 17 somites, before any apparent limb outgrowth had occurred, in
wild-type and ß-catPrx1/- limbs
(Fig. 2C,C'). At 20
somites, Fgf8 expression was still comparable in
ß-catPrx1/- and wild-type limbs
(Fig. 2D,D'). However, at
25 somites, before the morphological appearance of an AER, Fgf8
expression was reduced in a patchy manner in
ß-catPrx1/- limbs (see arrows in
Fig. 2E,E'). Shortly
after the formation of the stratified AER at stage E10.5, Fgf8
expression was restricted to patches along the distal margin in
ß-catPrx1/- limbs
(Fig. 2F,F'). The patchy
downregulation of Fgf8 in the AER and the subsequent loss of the AER
suggest a defect in AER maintenance upon loss of ß-catenin activity in
the mesenchyme.
ß-catenin had been implicated in the regulation of Tbx5 in
chick (Kawakami et al., 2001)
and Fgf10 in mouse (Agarwal et
al., 2003) during limb bud initiation. We therefore examined their
expression in ß-catPrx1/- limbs. Around 19-20
somites, the expression of Tbx5 and Fgf10 in
ß-catPrx1/- forelimbs was not altered
(Fig. 2G,G',I,I'),
suggesting either that these genes are not directly controlled by
ß-catenin or that the Prx1-Cre line does not delete early and broadly
enough to see an effect. However, at E10.75, Fgf10 expression was
downregulated (Fig.
2H,H'), although Tbx5 expression was still
maintained (Fig. 2J,J',
see also Fig. S3A-A'' in the supplementary material).
Overactivation of ß-catenin in the limb mesenchyme causes premature AER regression
Because Fgf8 expression and functional AER activity was
significantly affected in forelimbs lacking ß-catenin, we analysed
whether a gain of mesenchymal ß-catenin activity would result in the
ectopic expression of AER markers. Fgf8 was expressed ectopically in
distal patches in ß-catex3Prx1/+ forelimbs at
E10.5 (data not shown) and E11.5 (Fig.
3A', red arrow). However, this was not observed for other
AER markers, such as Fgf4 (Fig.
3G', E10.5), Fgf9
(Fig. 3H', E11.5) and
Wnt3 (data not shown). Fgf8 section in situ hybridizations
revealed that the ectopic expression was mesenchymal (see Fig. S3B' in
the supplementary material).
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ex3Prx1/+ forelimbs
from the posterior AER at E11.5 (Fig.
3A', white arrow). At E12.5, in comparison to wild-type
limbs, Fgf8 expression was either completely absent, or only
maintained in the most anterior AER (Fig.
3B'', data not shown),
(Fig. 3B). By contrast,
although no ectopic Fgf8 expression was detected in
ß-catex3Prx1/+ hindlimbs at E11.5
(Fig. 3A') or E12.5
(Fig. 3B'), Fgf8
expression was also lost from the posterior AER from E12.5 onwards
(Fig. 3B'). In order to
quantify the extent of AER loss, we measured the Fgf8 expressing
region in the fore- and hindlimbs of wild-type and
ß-catex3Prx1/+ embryos at E10.5 and E11.5
(Fig. 3C). No significant
differences in AER length were detected at E10.5 between wild type and
ß-catex3Prx1/+. However, at E11.5 there was a
significant reduction in both fore- (45.4%) and hindlimbs (7.9%) of
ß-catex3Prx1/+ embryos
(Fig. 3C). SEM analyses were
performed at E10.75 and E11.5. These revealed slight morphological differences
between wild-type (Fig. 3D,E)
and ß-catex3Prx1/+
(Fig. 3D',E')
forelimbs at E10.75, such as a flattened posterior AER (bracket) and
differences in the shape (arrowhead). At E11.5, the AER was only visible in
the anterior region of ß-catex3Prx1/+
forelimbs (Fig. 3F',
between arrowheads).
As other FGF molecules have been implicated in AER maintenance, we analyzed
Fgf4, Fgf9, Fgf17 and Fgf10 expression in
ß-catex3Prx1/+ limbs. Fgf4, expressed
in the posterior AER of wild-type forelimbs up to around E11.5, was severely
reduced in the posterior AER of ß-catex3Prx1/+
forelimbs at E10.5 (Fig.
3G'). Similarly, Fgf9 expression was also
prematurely lost in ß-catex3Prx1/+ forelimbs
at E11.5 (Fig. 3H,H').
Fgf17 was already downregulated in the AER of both wild-type and
ß-catex3Prx1/+ forelimbs around E10.5 (data
not shown). Mesenchymal expression of Fgf10 was not altered at early
stages, but was reduced in ß-catex3Prx1/+
forelimbs from E11 onwards (Fig.
3I,I'; and data not shown). The premature loss of AER
markers and Fgf10 in gain-of-function limbs suggests premature
regression of the AER.
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ex3Prx1/+ forelimbs starting at E11.0
(data not shown) and was nearly completely lost at E11.5
(Fig. 3J,J'). Similarly,
Ptc1, which serves a molecular readout for Shh signaling was highly
reduced in E11.5 ß-catex3Prx1/+ limbs
(Fig. 3K,K').
We analyzed whether the levels of Bmps and their target genes were changed
in ß-catex3Prx1/+ limbs, as Bmp activity has
been implicated in AER regression. At E11.5, Bmp4 and Bmp7
were upregulated throughout ß-catex3Prx1/+
fore- and hindlimbs (Fig.
4A',B',H, and data not shown), whereas Bmp2
was only upregulated distally (Fig.
4C'). All three Bmp genes were highly expressed in the most
posterior region, including the AER (Fig.
4A',B',C'). In addition, the Bmp antagonist
gremlin was upregulated (Fig.
4D'), as were the Bmp target genes Msx1 and
Msx2 in the distal limb (Fig.
4E',F'). Vibratome sections of whole-mount stained
embryos (Fig. 4G-G''), and
in situ hybridization on sections (Fig.
4H, and data not shown) demonstrated that the ectopic expression
was mesenchymal. Upregulation of Bmp genes and gremlin occurred earlier
(E10.5-E10.75) and more extensively in fore- than in hindlimbs (data not
shown), implying that the upregulation is probably dependent on the level of
activated ß-catenin, as Cre recombination occurred earlier in fore- than
in hindlimbs. Conversely, levels of Bmp2, Bmp4, Bmp7, gremlin,
Msx1 and Msx2 were reduced in
ß-catPrx1/- limbs at E11.5 (data not shown).
Our data suggest that the limb truncations in
ß-catex3Prx1/+ embryos are due to a premature
regression of the AER, caused by an upregulation of Bmp gene expression and
activation of Bmp signaling.
Increased cell death in the mesenchyme of ß-catenin mutant limbs
The AER defects observed in ß-catenin lof and gof limbs could also be
due to excessive cell death in the distal mesenchyme or AER. We therefore
performed TUNEL assays on transverse sections through
ß-catPrx1/- and
ß-catex3Prx1/+ limbs at different
developmental stages. At 25 somites, corresponding to the onset of
downregulation of Fgf8 expression, no significant difference in
apoptosis was observed (Fig.
5A'). However, more TUNEL-positive cells were detected in
the proximal mesenchyme of ß-catPrx1/-
forelimbs at E10 (Fig.
5B'). At E11.5, ß-catPrx1/-
hindlimbs showed an extensive area of cell death in the central core of the
limb bud (data not shown). In gof forelimbs, an increased number of
TUNEL-positive cells, predominately scattered throughout the proximal
mesenchyme, was detected at E11 (Fig.
5C'). At E11.5, apoptosis had further increased and was more
widespread, including distal regions (data not shown). The amount of cell
death in the AER was comparable in
ß-catex3Prx1/+ and wild-type limbs
(Fig. 5C,C'). The
temporal occurrence of apoptosis increase in the mesenchyme of gof forelimbs
corresponds to Bmp upregulation, suggesting that they may play a role.
However, gof hindlimbs, in which Bmps were also expressed ectopically, showed
no increase in TUNEL-positive cells at E11.5 (data not shown).
Lmx1b is regulated by ß-catenin in the limb mesenchyme
Dorsoventral limb patterning is determined by the correctly localized
expression of three genes: Wnt7a and En1, expressed in
dorsal and ventral ectoderm, respectively, and Lmx1b, which is
regulated by Wnt7a and expressed in the dorsal mesenchyme. At E10.5,
the ectodermal regulators of Lmx1b, Wnt7a
(Fig. 6A',A'') and
En1 (Fig.
6B',B'') were expressed in both ß-catenin
lof and gof limbs similar to wild type
(Fig. 6A,B). Loss of
ß-catenin activity resulted in reduced and partial loss of Lmx1b
expression in the forelimb already at E9.5
(Fig. 6C'), when compared
with wild type (Fig. 6C).
Conversely, in ß-catenin gof limbs, Lmx1b expression was
expanded into the ventral mesenchyme at E9.5
(Fig. 6D', black arrow)
compared with wild type (Fig.
6D). At E11.5, Lmx1b expression was maintained in
ß-catPrx1/- forelimbs, primarily in regions
adjacent to the remnants of a functional AER
(Fig. 6E'). Section in
situ hybridizations on E10.5 ß-catPrx1/-
forelimbs revealed an overall downregulation of Lmx1b and an even
more striking downregulation in the dorsal-most regions, primarily in the
distal half (Fig. 6F').
The expansion of Lmx1b into the ventral mesenchyme was clearly
visible on E10.5 ß-catex3Prx1/+ forelimb
sections. However, a few Lmx1b-negative patches were observed within
the dorsal and ventral mesenchyme (Fig.
6F''). These areas were not apoptotic, as confirmed by TUNEL
staining (data not shown). Notably, no ectopic Lmx1b expression
directly adjacent to the ventral ectoderm
(Fig. 6F'', black arrow)
was observed in any of the examined
ß-catex3Prx1/+ forelimb sections
(n=3). In the hindlimbs, small patches of cells expressing
Lmx1b adjoining the ventral ectoderm were occasionally detected
(n=1/3; data not shown).
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ex3Prx1/+ embryos suggested a
cell-autonomous effect on Lmx1b. To further investigate this, the
distribution of stabilized ß-catenin protein was compared with Lmx1b,
Lef1 and Tcf1 expression in
ß-catex3Prx1/+ hindlimbs on adjacent sections
at E11.5. This revealed high levels of ß-catenin protein throughout the
entire mesenchyme (black arrow in Fig.
6G',G''; compared with the trunk, white arrow),
intermingled with areas with lower levels
(Fig. 6G',G'';
arrowhead). The latter were probably the result of incomplete
Cre-recombination. Lmx1b expression always correlated with the
presence of stabilised ß-catenin (Fig.
6G',H'). However, in the ventral-proximal region,
Lmx1b was not upregulated, despite the presence of high levels of
stabilized ß-catenin (Fig.
6G',H' black arrows). The two known target genes and
transcriptional co-regulators of ß-catenin Lef1 and
Tcf1 (Hovanes et al.,
2001; Kengaku et al.,
1998) were also upregulated in areas with high levels of
ß-catenin (Fig.
6I'-J'). In contrast to Lmx1b, they were
upregulated in ventral-proximal regions (black arrows in
Fig. 6I',J'). Like
Lmx1b, Lef1 and Tcf1 expression were downregulated in
ß-catenin lof limbs (see Fig. S3D'',E'' in the supplementary
material). Double-fluorescent section in situ hybridization revealed that
Lmx1b-positive cells also upregulated Lef1 and Tcf1
(Fig. 6H'',I'',K, and
data not shown). Our results suggest that Lmx1b is probably
regulated, in part, by the canonical Wnt signaling pathway.
ß-catenin regulates scapula formation
Early limb patterning defects are often reflected in alterations of
internal structures, such as the skeletal elements. Whole-mount Alizarin
Red/Alcian Blue skeletal staining of E18.5 embryos revealed the presence of a
hypoplastic scapula in the ß-catPrx1/-
forelimb. This consisted only of a small remnant of, presumably, the scapula
blade (Fig. 7A',
arrowhead) and of a small, round element attached to the proximal end of an
element, which is likely to result from incomplete separation of the
humerus/radius/ulna anlage (Fig.
7A', arrow). Section in situ hybridizations on E12.5
forelimbs using probes for collagen 2
1 and Gdf5 showed that
part of the scapula anlage (Fig.
7B, arrowhead) was absent in
ß-catPrx1/- embryos
(Fig. 7B', arrowhead).
The Gdf5 staining showed that a separate element articulated with the
`humeral' end (Fig. 7D',
green arrow). At E14.5, this element (Fig.
7C, black arrow) appeared to be partially fused to the `humerus'
(Fig. 7C,E). This element
probably corresponds to the glenoid fossa that articulates with the
humerus.
As the skeletal elements of loss- and gain-of ß-catenin embryos were
either severely malformed or almost completely missing, respectively, we
analyzed various proximodistal markers, such as Hoxc6, Hoxa9, Hoxa10,
Meis1 and Meis2, at E10.5
(Fig. 7F-I''), to see
whether limb patterning was still normal
(Capdevila et al., 1999;
Favier et al., 1996;
Fromental-Ramain et al., 1996;
Mercader et al., 1999;
Nelson et al., 1996;
Wahba et al., 2001). The
expression of Hoxc6, normally expressed in the proximo-anterior
region (Fig. 7F), shifted
distally in ß-catPrx1/-
(Fig. 7F'), but was not
altered in ß-catex3Prx1/+ limbs
(Fig. 7F''). The Hox genes
Hoxa9 and Hoxa10, expressed in the distal regions of the
limb (Fig. 7G,H), remained
distally expressed in lof (Fig.
7G',H') and gof
(Fig. 7G'',H'')
limbs. The proximal markers Meis1 and Meis2
(Fig. 7I, and data not shown)
remained proximal in lof (Fig.
7I') and gof (Fig.
7I'') limbs.
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; Timmons et al.,
1994; Wilm et al.,
1998). In wild type, Pax1 and Emx2 were normally
expressed in the proximo-anterior forelimb mesenchyme at E10.5
(Fig. 7J,K), whereas their
expression was highly reduced in ß-catPrx1/-
forelimbs (Fig.
7J',K'). Section in situ hybridization revealed that
Emx2 was still expressed in a small patch of cells in the
anterior-proximal region of ß-catPrx1/- limbs
(data not shown).
In ß-catex3Prx1/+ limbs, the Pax1
domain was slightly reduced (Fig.
7J''), whereas Emx2 expression was upregulated
throughout the proximal limb at E11 (Fig.
7K''). These data suggest that Emx2, but not
Pax1, could be a direct target of canonical Wnt signaling in the
developing limb. Furthermore, agenesis of the scapula in lof ß-catenin
mutants is probably due to the downregulation of Emx2 and
Pax1 in ß-catPrx1/- mutant
embryos.
Stabilizing ß-catenin in vitro can upregulate Emx2 but not Lmx1b
Using an in vitro system, we assayed for expression changes of various
genes, including Bmp2, Bmp4, Bmp7, Lmx1b, Lef1, Tcf1, Emx2 and
Twist, which were upregulated in the
ß-catex3Prx1/+ limbs (see Figs
4,
6,
7, and Fig.
S3C',D',E' in the supplementary material). Semi-quantitative
RT-PCR analysis was performed on RNA from high-density micromass cultures from
E11 ex3fl/ex3fl embryos infected with an Adeno-Cre virus, resulting in the
accumulation of significant amounts of the N-terminal truncated stabilized
form of ß-catenin 8-10 hours after transfection
(Hill et al., 2005). Control
cultures were infected with an Adeno-Gfp virus. Bmp4, Bmp7, Lef1,
Tcf1 and Emx2 (Fig.
7L, see also Fig. S4 in the supplementary material) were
upregulated in this assay, similar to the in vivo observations. Surprisingly,
Lmx1b, and also Twist and Bmp2, were not
upregulated in vitro, unlike in vivo (Fig.
7L, Figs S3, S4 in the supplementary material). Like in vivo,
expression of Tbx5 and Fgf10 was not altered (see Fig. S4 in
the supplementary material). Sox9 was downregulated, as previously
shown (Fig. S4 in the supplementary material)
(Hill et al., 2005). The in
vitro results suggest that Tcf1, Lef1, Bmp4, Bmp7 and Emx2
could be direct targets of active ß-catenin signaling in the limb.
However, Lmx1b and also Twist, which cannot be upregulated
by ß-catenin in vitro, might be under the co-regulatory control of a
ß-catenin-mediated Wnt signal and another signal emanating from the
ectoderm in vivo.
|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Ng et al.,
2002). So far, no Wnt mutant in mouse has been described where
limb initiation failed. Similarly, limb initiation still occurs in
Lef1/Tcf1 double mutants; however, this might be due to the presence
of Tcf3 and Tcf4 (Agarwal
et al., 2003). By contrast, inactivation of Tbx5 activity
results in a complete absence of forelimbs or pectoral fins
(Agarwal et al., 2003;
Ahn et al., 2002;
Garrity et al., 2002;
Ng et al., 2002;
Rallis et al., 2003). Despite
the fact that we used the same Cre-deleter line (Prx1-Cre) as in a previous
study demonstrating that Tbx5 is essential for limb initiation
(Rallis et al., 2003), we
observed limb outgrowth and initially normal expression of Tbx5,
Fgf10 and Fgf8. This would be in agreement with the previously
suggested model that Tbx5 acts upstream of Wnt/Fgf signaling
(Agarwal et al., 2003;
Takeuchi et al., 2003).
However, the Prx1-Cre line is probably not suitable to address the question of
whether canonical Wnt/ß-catenin is required for Tbx5 initiation,
as Tbx5 expression is observed at 8 somites (E8.5)
(Agarwal et al., 2003), whereas
Prx1-Cre was active only from 15 somites onwards (see Fig. S2 in the
supplementary material).
Contrasting with a previous in vitro observation showing that
ß-catenin can activate a Fgf10 promoter construct
(Agarwal et al., 2003), our
results suggest that Fgf10 is not regulated by Wnt/ß-catenin
signaling. The observed Fgf10 downregulation is probably due to a
disruption in the Fgf8/Fgf10-feedback loop, as Fgf8
expression in the AER is clearly reduced prior to Fgf10
expression.
Our data show that mesenchymal ß-catenin, together with ectodermal
Wnt/ß-catenin signaling, is required for Fgf8 expression and AER
maintenance (Barrow et al.,
2003; Soshnikova et al.,
2003); however, the underlying molecular mechanism needs to be
further investigated, as Fgf10 does not seem to be the direct target.
Possible targets could be brachyury or Twist, which are regulated by
canonical Wnt/ß-catenin signaling in other tissues
(Galceran et al., 2001;
Howe et al., 2003;
Yamaguchi et al., 1999b). They
are expressed in the distal mesenchyme and have previously been implicated in
AER maintenance (Liu et al.,
2003; Zuniga et al.,
2002). Twist is the most likely candidate, as it was
upregulated in gain-, and downregulated in loss-of-function ß-catenin
limbs (see Fig.
3C',C'').
|
ex3Prx1/+ limbs is due to premature
regression of the AER starting posteriorly, whereby loss of Fgf4 and
Fgf9 preceded that of Fgf8. Downregulation of Shh
coincided with the loss of Fgf4 and Fgf9, suggesting that
the regression is caused by a disruption in the ZPA/AER-feedback loop
(Sun et al., 2000). The
molecular mechanism underlying the observed AER regression is probably the
ß-catenin-mediated upregulation of Bmp genes, as BMPs can induce AER
regression (Guha et al., 2002;
Khokha et al., 2003;
Pizette and Niswander, 1999;
Zuniga et al., 1999). This
activity is normally antagonized by gremlin
(Khokha et al., 2003). Gremlin
was also upregulated upon activation of ß-catenin; however, we think that
this is a secondary effect, because concurrent expansion of the Bmp target
genes Msx1 and Msx2 suggests that active Bmp signaling was
occurring in ß-catex3Prx1/+ limbs, and because
Bmps can induce gremlin expression
(Pereira et al., 2000).
It is interesting to note that Wnt5a, which is expressed in the
distal limb mesenchyme, has recently been shown to downregulate ß-catenin
levels (Topol et al., 2003).
Wnt5a-/- embryos show a suppression of Sox9
expression and chondrogenesis in the distal structures, resembling the
phenotype of ß-catex3Prx1/+
(Hill et al., 2005;
Topol et al., 2003). However,
no AER defects have been reported and the expression of Bmp genes was not
analyzed in these limbs (Yamaguchi et al.,
1999a). These discrepancies could be due to differences in the
level or timing of ß-catenin stabilization between
ß-catex3Prx1/+ and
Wnt5a-/- embryos. It would certainly be interesting to see
which aspects of the Wnt5a limb phenotypes could be rescued by
removing ß-catenin. Proper limb development probably requires the
fine-tuning of ß-catenin levels in the distal mesenchyme through the
Wnt5a destabilizing activity and the stabilizing activity of
canonical Wnts from the AER.
Lmx1b expression is controlled by ß-catenin signaling
Lmx1b expression has been shown to be regulated by Wnt7a
in chicken and mouse (Cygan et al.,
1997; Loomis et al.,
1998; Riddle et al.,
1995; Vogel et al.,
1995); however, it is probably not mediated by the canonical
ß-catenin pathway (Kengaku et al.,
1998). Surprisingly, we found that the loss of ß-catenin
activity in mice resulted in a downregulation of Lmx1b as early as
E9.5 in the dorsal mesenchyme. Concomitantly, overactivation of ß-catenin
resulted in a cell-autonomous upregulation of Lmx1b; however, not all
regions of the limb were capable of responding. This, together with the
observation that Lmx1b expression could not be upregulated in vitro,
unlike a number of other genes that were also upregulated in vivo, suggests
that Lmx1b regulation by ß-catenin requires an additional
signaling input.
Despite the recent data showing that Wnt7a signaling in the presence of
Lrp6 stabilizes ß-catenin (Adamska et
al., 2005; Caricasole et al.,
2003), we do not think that Wnt7a uses the canonical
pathway to regulate Lmx1b. Especially because Lmx1b
expression is only downregulated in the distal mesenchyme of Wnt7a
and Lrp6 knockout mice at E11.5 and E12.5, respectively
(Adamska et al., 2005;
Cygan et al., 1997), whereas
in ß-catPrx1/- limbs it is reduced as early as
E9.5. Lmx1b is also upregulated in
ß-catex3Prx1/+ limbs around this stage. By
contrast, ectopic Wnt7a expression in En1-/-
limbs from E9.5 onwards leads to ventral expansion of Lmx1b only at
E11.0 (Cygan et al., 1997;
Loomis et al., 1998).
Therefore, we favor the hypothesis that, in addition to Wnt7a, one or
more Wnt(s) are involved in Lmx1b regulation, and that these other
Wnt(s) signal through the canonical pathway. Possible candidates would be
Wnt3, Wnt4, Wnt6 and Wnt7b, which are expressed in the
ectoderm (Bennett et al.,
2002). However, these Wnts are expressed throughout the limb bud
ectoderm and therefore should activate canonical Wnt/ß-catenin signaling
in both dorsal and ventral mesenchyme adjacent to the ectoderm. This would
correspond to the observed high levels of ß-catenin protein, and the
expression of Tcf1 and Lef1 in the subectodermal mesenchyme
(Fig. 6). Therefore, we propose
that ß-catenin-dependent Lmx1b expression requires probably an
additional factor, emanating from the dorsal ectoderm or restricted to the
dorsal mesenchyme. This is in agreement with the observation that the in vitro
stabilization of ß-catenin was insufficient to upregulate Lmx1b.
Alternatively, a secreted repressor of Lmx1b may be present in the
ventral ectoderm, which could explain why ectopic Lmx1b expression
was never observed in the ventral-most mesenchyme. Based on our results, we
favor a combination of both mechanisms in order to restrict Lmx1b to
the dorsal mesenchyme. Hence, the ventral ectoderm might play a dual role in
preventing Lmx1b activation in the ventral mesenchyme: by expressing
En1, which inhibits Wnt7a, and by the expression of a
putative secreted inhibitor interfering specifically with
ß-catenin-mediated activation of Lmx1b. Interestingly, loss of
ß-catenin in the ectoderm also affects dorsoventral patterning, but this
is due to loss of En1 (Barrow et
al., 2003; Soshnikova et al.,
2003).
ß-Catenin is required for scapula development
ß-catPrx1/- embryos display severe agenesis
of the scapula. Although the process of skeletogenesis is closely connected to
that of limb patterning, scapula agenesis in mice lacking ß-catenin in
the limb mesenchyme is probably not caused by the changes observed in skeletal
differentiation (Hill et al.,
2005). Lack of ß-catenin does not inhibit chondrocyte
differentiation, on the contrary it stimulates it
(Day et al., 2005;
Hill et al., 2005). However,
in ß-catPrx1/- limbs the cartilaginous scapula
anlage is not formed (Fig.
7B'), resembling the Emx2-/- phenotype
(Pellegrini et al., 2001).
Concomitantly, Emx2, but also the transcription factor Pax1,
which is important for scapula formation as well, are severely downregulated
(Pellegrini et al., 2001;
Timmons et al., 1994;
Wilm et al., 1998). The loss
of these two transcription factors during early limb bud development is
probably the cause for scapula agenesis. ß-catenin seems to be necessary
and sufficient for Emx2 expression, making Emx2 a potential
direct target of canonical Wnt/ß-catenin signaling in the limb
mesenchyme, as has been previously shown in the telencephalon
(Backman et al., 2005;
Theil et al., 2002). The
observed increase in cell death in the proximal region of
ß-catPrx1/- limbs at E10 could in principal
contribute to scapula agenesis; however, a similar pattern of cell death is
observed in Fgf4/Fgf8 double mutants, and even more
extensive cell death is found in Shh and gremlin mutant limbs, but
those develop a normal scapula (Boulet et
al., 2004; Michos et al.,
2004; Sun et al.,
2002; te Welscher et al.,
2002).
The loss of the scapula, along with the other appendicular skeletal
elements, in ß-catex3Prx1/+ forelimbs is due
to the fact that stabilization of ß-catenin results in a complete block
of skeletogenesis, as has been shown previously
(Hill et al., 2005), and is
likely to be independent of the changes in Emx2 and Pax1
expression.
Our results provide strong genetic evidence that, in the limb mesenchyme, ß-catenin activity is required for AER maintenance, and also, eventually, for its regression, via the control of Bmps. Furthermore, mesenchymal ß-catenin signaling is required for dorsal mesenchyme identity through regulation of the dorsal selector gene Lmx1b, and, in addition, is required for scapulagenesis, principally via the regulation of Emx2.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/7/1219/DC1
|
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