. Wnt3a-regulated genes were
identified using SAM (Tusher et al.,
2001
). Chick cells were cultured overnight, then stimulated with
250 ng/ml Wnt3a and/or 150 ng/ml Fgf8b. Total RNA was isolated and reverse
transcribed using RNeasy (Qiagen) and Thermoscript II (Invitrogen) kits and
random hexamer primers. Real-time PCR was performed using a Roche Lightcycler
and FastStart DNA Masterplus SYBR Green I reagents (Roche). Primer
sequences are available upon request.
Mutant animals
Prx1::Nmyc transgenic mice were generated by zygote microinjection
of a DNA construct containing the Prx1 XB2.4 promoter/enhancer
(Martin and Olson, 2000)
driving a rabbit β-globin intron, an N-terminally FLAG-tagged
mouse Nmyc-coding sequence, and a rabbit β-globin polyA
signal. Nmyc-/- mutant mice (Mycn-/- -
Mouse Genome Informatics) are described elsewhere
(Knoepfler et al., 2002).
Detection of BrdU, gene expression and reporter activity
Embryos were labeled for 1 hour by intraperitoneal injection of the mother
with 50 mg/kg BrdU (Sigma). Tissues were fixed with Bouin's fluid (Sigma) for
Sdc1 staining, 4% paraformaldehyde for Col1 in situ hybridization, and
Histochoice MB (Amresco) for other immunohistochemistry and in situ
hybridization. Monoclonal 
-BrdU (Sigma, 1:1,000), -myosin heavy
chain MF20 and -procollagen 1 M-38 (Developmental Studies Hybridoma
Bank, 1:100), rat monoclonal -Syndecan-1 (BD Pharmingen, 25 µg/ml)
and rabbit -Sox9 (Santa Cruz Biotechnology, 1:100) antibodies were
detected using Vectastain elite ABC kit and DAB (Vector Labs), or via
fluorescence using biotinylated secondary antibodies and Cy3- or
Alexa488-streptavidin. For MF20/M38 double staining, cryosections were stained
with MF20 and detected using Alexa488-streptavidin, blocked with avidin and
biotin, stained with M-38 and detected using Cy3-streptavidin. For in situ
hybridization, DIG-labeled probes were detected with NBT (Roche) for Col1
(Metsaranta et al., 1991), or
TSA amplified and detected with DAB using a GenPoint kit (DAKO, Nmyc). BrdU
density around beads was determined by counting BrdU-positive nuclei in
50x50 µm squares around the beads in every third 7 µm section. At
least 20 squares per bead were counted. lacZ was detected in whole
mounts (E9.5) or cryosections (E10.5 and E11.5) by X-gal staining, and
sections counterstained with nuclear Fast Red (Vector Labs).
Adenovirus infection and proliferation assays
Limb bud cells plated in 96-well plates at 5x104
cells/well were infected with 5x105 pfu/well of adenovirus
(Kuhnert et al., 2004) for 12
hours. The cells were cultured an additional 12 hours followed by labeling
with 10 µm BrdU for 4 hours. Cells were then trypsinized, split over 2
plates and assayed for BrdU (Cell proliferation ELISA, Roche) and total DNA
content (Cyquant cell proliferation assay, Invitrogen). BrdU values were
normalized using the DNA values. Unpaired two-tailed t-test or
one-way ANOVA, as indicated, was performed to determine significance. Results
shown are the mean±s.e.m.
 |
RESULTS
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Limb ectoderm inhibits chondrogenesis and promotes proliferation of limb mesenchyme via Wnt signals
Limb ectoderm inhibits chondrogenesis (Kosher, 1979;
Solursh et al., 1981), and we
tested whether this was mediated by Wnt signals. We visualized Wnt signaling
using cells derived from Axin2lacZ/+ mutant mice, in which a
lacZ reporter gene has been inserted into the Wnt target gene Axin2
(Aulehla et al., 2003;
Jho et al., 2002;
Lustig et al., 2002). Thus,
X-gal staining indicates Wnt responsiveness. Wild-type limb ectoderm cultured
on top of Axin2lacZ/+ limb mesenchyme induced the Wnt reporter
(Fig. 1A), and inhibited
chondrogenesis in the mesenchymal cells around it
(Fig. 1B). In the presence of
the Wnt antagonist Fz8CRD (Hsieh et al.,
1999), Wnt reporter activity was markedly reduced
(Fig. 1C) and chondrogenesis
occurred (Fig. 1D). These data
indicate that limb mesenchyme responds to a Wnt signal from the ectoderm and
that this signal inhibits chondrogenesis.
Several studies have shown that genetic activation of the Wnt pathway
inhibits chondrogenesis (Hartmann and
Tabin, 2000; Rudnicki and
Brown, 1997). We tested here whether purified Wnt3a protein was
able to do this. In culture, limb mesenchyme responded to Wnt3a protein by
induction of the reporter (Fig.
1E,F) and chondrogenesis in these cells was inhibited
(Fig. 1G,H). To confirm that
Wnt signals were able to inhibit chondrogenesis in vivo as well, we implanted
Wnt3a beads into developing limb buds. Wnt3a beads induced reporter activity
(Fig. 1I,J) and the protein
alone was sufficient to block chondrogenic differentiation in cells around the
Wnt source (Fig. 1K,L).
Combined, these results demonstrate that Wnts are necessary and sufficient for
the chondroinhibitory effect of limb ectoderm.
Cell proliferation in the limb bud is associated with the presence of
nearby ectoderm
(Fernández-Terán et al.,
2006; Janners and Searls,
1970; Köhler et al.,
2005), and we next tested whether limb ectoderm had
proliferation-inducing activity. Indeed, ectoderm cultured on top of limb
mesenchyme induced proliferation as measured by BrdU incorporation
(Fig. 1M). Moreover, this
effect depends on Wnt signals as it was abolished by the Wnt antagonist Fz8CRD
(Fig. 1N). To test whether Wnt
signals were sufficient to promote proliferation in vivo, we implanted Wnt3a
beads into limb buds and assayed for proliferation using BrdU labeling.
Whereas vehicle beads had no effect (Fig.
1O), a strong increase in BrdU labeling was observed around Wnt3a
beads (Fig. 1P). Combined, our
data show that the limb ectoderm, by secreting Wnts, not only inhibits
chondrogenic differentiation but also promotes proliferation in the underlying
mesenchyme.
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Fig. 1. Limb ectoderm inhibits chondrogenesis and promotes proliferation via Wnt
signals. (A) Chick limb ectoderm cultured on top of E11.5
Axin2lacZ/+ limb mesoderm cells induces the reporter in
the mesoderm (n=10). (B) Chick limb ectoderm cultured on top
of chick limb mesoderm inhibits chondrogenesis (stained with Alcian Blue) in a
 150 µm wide zone around the explant (n=30). (C) The
Wnt antagonist Fz8CRD inhibits induction of the
Axin2lacZ/+ reporter by the ectoderm (n=10). The
ectoderm in A and C is derived from stage 24 chick limb buds and does not
carry the reporter. (D) Fz8CRD protein abrogates the chondro-inhibitory
effect of the ectoderm (45 out of 48). (E,F) E11.5
Axin2lacZ/+ limb bud cells induce the lacZ
reporter (blue) in response to Wnt3a protein (100 ng/ml for 19 hours; F).
(G,H) Chick stage 22 limb mesenchyme cells undergo
chondrogenesis (stained with Alcian Blue) in micromass cultures (G), which is
inhibited by 100 ng/ml Wnt3a protein (H, n>200).
(I,J) Activation of the reporter by Wnt3a beads (arrowheads, J)
but not vehicle beads (I) implanted in E11.5 Axin2lacZ/+
limb buds (n=4). (K,L) Vehicle beads implanted in stage
22 wing buds become embedded in the cartilage of the humerus (K), whereas
chondrogenesis (stained with Alcian Blue) is inhibited around Wnt3a beads (L,
n=8). (M,N) Chick limb ectoderm cultured on top of
chick limb mesoderm induces BrdU incorporation (blue) in the surrounding cells
(M, n=17), which is inhibited by Fz8CRD (N, n=18; nuclei
labeled in red). (O,P) Sections through limb buds cultured with
implanted vehicle (O) or Wnt3a (P) beads. Wnt3a induces BrdU incorporation
around the bead (n=11). Scale bars: 100 µm in A-D,M-P; 500 µm
in E-H; 200 µm in I-L. h, humerus; r, radius; u, ulna; B, bead; E,
ectoderm.
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Wnt signals re-specify limb progenitors from cartilage towards soft connective tissue fates
We next addressed whether Wnt3a maintained limb mesenchyme in an
undifferentiated state or allowed differentiation into other tissues. We first
tested whether Wnt3a maintained the chondrogenic potency of the cells, which
would indicate that they remained undifferentiated. Although the cells
remained chondrogenic following short exposures to Wnt3a, they lost their
chondrogenic potency following prolonged (>42 hours) exposure (see Fig. S1A
in the supplementary material; Fig.
2A,B), suggesting that they differentiated into other tissue
types. During limb development, the connective tissues that envelop the
chondrogenic core form in the vicinity of the Wnt-producing ectoderm. The
ectodermal Wnt signal might therefore change cell type specification from
chondrogenic towards soft connective tissues. To test this, we cultured cells
in the presence of Wnt3a and monitored the expression of a panel of
differentiated tissue markers (Table
1). Over the course of 8 days, the cells upregulated expression of
collagen 1, tenascin C, decorin, Dermo1 and Bmp3
(Fig. 2C), whereas expression
of scleraxis or the (pre)osteoblast marker osteopontin was not detected
(Table 1). This combination of
markers suggested differentiation towards soft connective tissue, specifically
perichondrium or perhaps dermis. These tissues are indeed Wnt responsive in
vivo, as demonstrated by Axin2lacZ/+ expression in perichondrium
and dermis of an E13.5 limb bud (see Fig. S1B in the supplementary
material).
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Fig. 2. Wnt3a protein re-specifies chondrogenic cells towards soft connective
tissues. (A,B) Micromass cultures from chick limb mesenchyme
cells treated with vehicle (A) or Wnt3a (B) for 4 days, cultured another 4
days in absence of Wnt3a. Wnt3a-treated cells failed to undergo chondrogenesis
(Alcian Blue; n>50). (C) Collagen 1, tenascin C, decorin,
Dermo1 and Bmp3 expression levels in high density cultures grown in continuous
presence of Wnt3a (blue), or during the first 3 days of culture, after which
the Wnt3a was replaced by vehicle (red). Time points 1 and 8 days were sampled
twice (mean±s.e.m.). (D) Small numbers of myotubules,
immunostained for myosin heavy chain (brown), accumulate in the periphery of
6-day-old mouse limb mesenchyme micromass cultures (n=6). (E)
Continuous treatment with Wnt3a (250 µg/ml) slightly expands the myotubule
number (n=6). (F) When Wnt3a is removed after 3 days of
culture, large numbers of myotubules spread over the tissue layer
(n=6). (G) Section through a chick wing 3 days after
implantation of a vehicle bead at embryonic stage 22, showing the bead
embedded in cartilage (n=14, Safranin O). (H) Wnt3a beads are
never embedded in cartilage (n=14) but surrounded by ectopic muscle
fibers, visualized by myosin heavy chain immunostaining (brown, n=7).
(I,J) Section through a control chick wing (I) and a wing with
implanted Wnt3a bead (J), immunostained for pro-collagen 1 (red) and myosin
heavy chain (green), nuclei stained blue (DAPI) (n=4). Both sections
are at a similar location and plane. Scale bars: 500 µm in A,B; 100 µm
in G-J. B, bead; car, cartilage.
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Transient exposure to Wnt3a changed the type of connective tissue that was
formed: following withdrawal of Wnt3a after 3 days of culture, collagen 1,
decorin and, to a lesser extent, tenascin C continued to be up regulated;
scleraxis and osteopontin remained undetectable, again suggesting
differentiation towards soft connective tissue
(Fig. 2C). But as Dermo1 and
Bmp3 were not induced (Fig.
2C), the combination of markers suggested differentiation towards
muscle connective tissue. This is consistent with a previous report showing
that in vivo activation of the Wnt signal transducer β-catenin induces
formation of ectopic muscle connective tissue
(Kardon et al., 2003). In
vivo, muscle connective tissue controls muscle differentiation
(Chevallier and Kieny, 1982;
Chiquet et al., 1981;
Kardon, 1998;
Kieny and Chevallier, 1979),
and we tested whether this occurred in vitro as well. We therefore established
micromass cultures from whole limb bud mesenchyme, which includes myoblasts,
and cultured the cells for 6 days. Indeed, transient exposure to Wnt3a
strongly promoted the formation of myotubules in micromass cultures
(Fig. 2D,F). Continuous
exposure to Wnt3a protein, which does not promote formation of muscle
connective tissue, led to a small increase in the number of myotubules
(Fig. 2D,E), and it is possible
that Wnt signals also promote the proliferation of muscle progenitors
(Anakwe et al., 2003;
Geetha-Loganathan et al.,
2005). So far, our in vitro data suggest that Wnt signals promote
the formation of specific types of connective tissue, which in turn influences
myogenesis.
To confirm that Wnt3a re-specifies limb progenitors away from a
chondrogenic and towards a soft connective tissue fate in vivo, we implanted
Wnt3a beads into stage 22 chick wing buds. Vehicle beads became incorporated
into cartilage, and no disruption to tissue patterning or cell differentiation
was observed (Fig. 2G and data
not shown). By contrast, Wnt3a beads were never in contact with cartilage
(Fig. 2H, and see
Fig. 1L) but disrupted the
pattern of cartilage differentiation and that of muscle and connective tissue
(Fig. 2H). Ectopic bundles of
muscle fibers were aligned around the Wnt3a beads, and in all cases there was
a layer of non-muscle tissue between the ectopic muscle and the beads
(Fig. 2H). Combined staining
for muscle fibers and pro-collagen 1-positive connective tissue demonstrated
that this was ectopic connective tissue
(Fig. 2I,J). Although we have
no data regarding the duration for which the beads provide active Wnt3a
protein, the stimulus can only be transient and would therefore promote the
formation of muscle connective tissue. In combination, our in vitro and in
vivo data suggest that Wnt signals re-specify limb progenitors from a
chondrogenic towards a soft connective tissue fate. The duration of Wnt
exposure influences which type of connective tissue forms, which in turn
controls the pattern of myogenesis.
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Fig. 3. Wnt and FGF proteins act in synergy to promote proliferation and
maintain the undifferentiated state. (A-G) Alcian Blue staining.
(A) Fgf8 protein delays chondrogenesis in micromass cultures of chick
stage 22-23 limb mesenchyme (n=12). (B) Wnt3a combined with
Fgf8 blocks chondrogenesis (n=12). (C) Removal of Fgf8 at day
4 of culture has little effect on chondrogenesis (n=12). (D)
Cells treated with Wnt3a and Fgf8 resume chondrogenesis upon removal of the
factors at day 4 (n=12). (E-G) Limb mesenchyme cells were
expanded for 4 days in presence of Wnt3a alone (E) or in combination with Fgf8
(F,G), trypsinized and replated as micromass cultures. Cells expanded in Wnt3a
alone lost their chondrogenic potential (E). Cells expanded in Wnt3a and Fgf8
retained their chondrogenic potential (F), whereas Wnt3a was still able to
inhibit their chondrogenesis (G) (n=4). (H) Wnt3a promotes
proliferation of limb mesenchyme in micromass cultures, which is enhanced by
Fgf8. Fgf8 alone does not enhance proliferation (n=4). (I)
Size of limbs cultured 4 days with intact ectoderm, or without ectoderm in
presence of the indicated factors (n=8, mean±s.e.m.).
(J-N) Representative examples of Alcian Blue-stained limb buds cultured
without ectoderm in presence of vehicle (J), Wnt3a (K), Fgf8 (L), Wnt3a and
Fgf8 (M), or with ectoderm left intact (N). Scale bar: 500 µm in J-N.
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Wnt and FGF signals combine to maintain limb progenitor cells in an undifferentiated state that retains the ability to undergo chondrogenesis
Our data show that Wnt signals control the segregation of multipotent
progenitor cells into chondrogenic and connective tissue lineages. The
multipotent progenitors themselves originate from the subridge region
(Pearse et al., 2007), but
what prevents their differentiation in this region? Cells in the subridge are
exposed to FGFs from the AER, in addition to Wnts from the ectoderm and AER.
We therefore tested whether FGF signals, alone or in combination with Wnt
signals, inhibited differentiation. Fgf8 protein delayed, but did not prevent,
chondrogenesis in micromass cultures (Fig.
3A,C), whereas the combination of Fgf8 with Wnt3a inhibited
chondrogenesis altogether (Fig.
3B). But in contrast to the effect of Wnt3a alone, the combination
of Fgf8 and Wnt3a maintained the undifferentiated state of the cells:
following withdrawal of both factors, they retained their ability to
differentiate into cartilage (Fig.
3D).
We then cultured limb mesenchyme cells for 4 days at high density in the
presence of Wnt3a alone, or in combination with Fgf8, and established
secondary micromass cultures. As expected, these micromasses were
non-chondrogenic when derived from cells expanded in presence of Wnt3a alone,
as they have switched to soft connective tissue fates
(Fig. 3E). By contrast, when
derived from cells expanded in presence of Wnt3a and Fgf8, the micromasses
differentiated into cartilage, similar to freshly isolated limb mesenchyme
(Fig. 3F). Moreover, Wnt3a was
still able to inhibit this chondrogenesis
(Fig. 3G), indicating that the
secondary micromasses remained responsive to developmental signals and
retained their multipotency.
Wnt and FGF signals combine to synergistically promote proliferation
We next tested whether FGF signals, alone or in combination with Wnt
signals, contribute to the proliferation of limb progenitors. Whereas Wnt3a
promoted growth in micromass cultures, Fgf8 protein alone was ineffective
(Fig. 3H). However, Fgf8
enhanced the proliferative effect of Wnt3a
(Fig. 3H). We observed the same
phenomenon in cultures of whole limb buds from which ectoderm and AER had been
removed: Fgf8 had little effect on growth, whereas the combination of Fgf8 and
Wnt3a strongly promoted growth, to a level on par with that of limb buds
cultured with intact ectoderm and AER (Fig.
3I-N). Alcian Blue staining revealed that the extra tissue was
largely of a chondrogenic nature (Fig.
3J-N), confirming that Wnt3a and Fgf8 promoted growth of
progenitors with a chondrogenic potential. Combined, our data show that the
combination of Wnt and FGF signals strongly promotes growth of limb progenitor
cells, while maintaining their undifferentiated multipotent state.
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Fig. 4. Regulation of target genes by Wnt3a and Fgf8 in chick limb
mesenchyme. Cells were cultured at high density in the presence of Wnt3a,
Fgf8, or both, and samples taken 2, 4 or 6 hours after addition of the
factors. Gene expression levels were plotted relative to vehicle controls.
Note synergistic regulation of Nmyc, Sdc1 and Sox9 by the
combination of Wnt3a and Fgf8 (blue line), and the antagonistic effect of Fgf8
on the induction of Nbl1 by Wnt3a.
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Synergistic and antagonistic regulation of target genes by Wnt and FGF
To determine which genes mediate the effects of Wnt3a and Fgf8 in limb
progenitor cells, we performed microarray analysis on E11.5 limb bud cells
treated with Wnt3a. The candidate target genes were then tested by real-time
PCR analysis of chicken stage 22-23 limb mesenchyme treated with Wnt3a. From
these two experiments, we identified a set of genes whose regulation by Wnt3a
was conserved between mouse and chick. In addition, we studied the response of
these genes to Fgf8, and to the combination of Wnt3a and Fgf8. Surprisingly,
more than half of the Wnt3a targets were also regulated by Fgf8, in some cases
synergistically and in other cases, in an antagonistic fashion.
The target genes fell into five categories
(Fig. 4): four genes were
induced by Wnt3a only (Apcdd1/Drapc1, Msx1,
Sostdc1/WISE/ectodin and Axin2); one gene was induced
by Fgf8 only [Dusp6/Mkp3, a known FGF target included for
comparison (Eblaghie et al.,
2003; Kawakami et al.,
2003; Pascoal et al.,
2007)]; two genes were induced synergistically by Wnt3a and Fgf8
(Nmyc and syndecan1/Sdc1); one gene was induced by Wnt3a but
this induction was antagonized by Fgf8 (Nbl1/DAN); and one
gene was repressed synergistically by Wnt3a and Fgf8 (Sox9).
We next examined whether the expression domains of these target genes were
consistent with their regulation by Wnts and FGFs, and with the temporal and
spatial distribution of cell behaviors (i.e. proliferation and cell fate
specification) within the limb bud. In both E10.5 and E11.5 limb buds, the
Axin2 reporter was expressed in 100 µm layer of mesenchyme underneath
the ectoderm in all regions of the limb bud
(Fig. 5A-C), consistent with
the expression of several Wnts in limb ectoderm
(Barrow et al., 2003;
Geetha-Loganathan et al.,
2005; Parr et al.,
1993; Roelink and Nusse,
1991). Apcdd1, another gene induced by Wnt alone
(Fig. 4), is similarly
expressed (Jukkola et al.,
2004).
Our data show that Wnt stimulates proliferation
(Fig. 1O,P) and BrdU labeling
indeed occurred predominantly in the Wnt-responsive region of the limb
(Fig. 5D-F). Wnt3a and Fgf8
synergize in promoting proliferation in culture
(Fig. 3H-N) and we found that
proliferation is highest in the subridge region where Wnt and FGF signals
overlap (Fig. 5D-F)
(Pascoal et al., 2007). A
similar pattern of expression was displayed by Nmyc and Sdc1
(Fig. 5J-M)
(Sawai et al., 1993;
Solursh et al., 1990), in
accordance with their synergistic induction by Wnt and FGF signals
(Fig. 4).
Wnt signals inhibit cartilage differentiation
(Fig. 1G,H,K,L). Consistent
with this, expression of the chondrogenic marker Sox9
(Bi et al., 1999) was limited
to the center of the limb bud, and absent from the region of Wnt signaling and
high cell proliferation (Fig.
5G-I). Nbl1 is induced by Wnt3a, and this induction is
antagonized by Fgf8 in vitro (Fig.
4); in vivo, Nbl1 is indeed expressed in peripheral
mesenchyme and excluded from the subridge region
(Pearce et al., 1999).
Although we classified Axin2 as a Wnt-only target, its induction by
Wnt3a is to some extent antagonized by Fgf8
(Fig. 4). In contrast to
Nbl1, Axin2 is expressed underneath the AER, although slightly weaker
ventrally, suggesting that this antagonism is not strong enough to overcome
the inducing signal (Fig.
5A-C). As Axin2 is an inhibitor of Wnt signaling, FGF might
stimulate the response to Wnt signaling by repressing Axin2.
Thus, we have identified target genes that are indicative of the presence,
the absence, or the overlap of Wnt and FGF signals. Moreover, the Wnt and
FGF-mediated cell behaviors (e.g. proliferation, differentiation,
multipotency) predicted from our in vitro analyses occur within the expression
domains of these genes in the limb bud. We observed these relationships
between cell behaviors and gene expression domains throughout the E10.5 limb,
but only in the distal half of the E11.5 forelimb
(Fig. 5B,E,H,K). This suggests
that at E11.5, subsequent patterning mechanisms come into operation in the
proximal limb to refine the patterns set up earlier by Wnts and FGFs.
Wnt promotes proliferation via Nmyc and inhibits chondrogenic differentiation via repression of Sox9
One of the target genes we found, Nmyc, is a member of the
myc family of oncogenes that mediate cell cycle entry in response to
proliferative signals (Trumpp et al.,
2001). Loss of Nmyc reduces proliferation and impairs
limb outgrowth starting at day E10.5
(Charron et al., 1992;
Ota et al., 2007;
Sawai et al., 1993;
Stanton et al., 1992). In situ
hybridization confirmed that Nmyc expression colocalized to the zone
of proliferating cells in the limb (Fig.
5D-F,J-L) and in the absence of Nmyc, cell division in
this zone was dramatically reduced (Fig.
6A,B). Nmyc also stimulates cell proliferation, as shown by viral
overexpression of the gene in limb mesenchyme and comparing the incorporation
of BrdU relative to a lacZ viral control
(Fig. 6C, one-way ANOVA,
P=0.0141).
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Fig. 5. Expression patterns of Wnt and FGF target genes correlate with cell
behaviors in mouse limb buds. (A-C) Expression of
Axin2lacZ/+ reporter, (D-F) BrdU labeling,
(G-I) Sox9 immunostaining, (J-L) Nmyc in situ
hybridization, (M) Sdc1 fluorescent immunostaining. Sections through
E10.5 forelimb buds (A,D,G,J), through the level of the central metacarpal in
E11.5 forelimb buds (B,E,H,K,M), through E11.5 hind limb buds (C,F,I,L). Note
co-localization of proliferation (BrdU labeling, D-F) with the
Axin2lacZ/+ reporter (A-C) and Nmyc expression
(J-L), whereas chondrogenic differentiation (Sox9 expression, G-I) is mutually
exclusive with proliferation and reporter expression. Dorsal is upwards,
distal is rightwards. Scale bars: 100 µm.
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To determine whether Nmyc was required for Wnt-induced
proliferation, we impaired Nmyc function by overexpressing a dominant-negative
form of the gene, Nmyc
MB2
(MacGregor et al., 1996;
McMahon et al., 2000), in
E11.5 limb bud cells. Wnt3a increased BrdU incorporation in control infected
cells by 46% (±13%, P=0.0090), which was significantly reduced
in NmycMB2-overexpressing cells (P=0.1809)
(Fig. 6C). This reduction is on
par with the reduction in cell proliferation achieved using the
cell-autonomous negative regulator of the Wnt pathway, Axin
(Zeng et al., 1997)
(P=0.0820) (Fig. 6C).
The remaining proliferation is probably from cells that resisted infection
(25% of the cells, not shown).
Following a second strategy to demonstrate that Wnt-mediated cell
proliferation depends upon Nmyc, we implanted Wnt3a beads into
Nmyc-/- limb buds and found that the extensive cell
proliferation previously observed was abrogated
(Fig. 6D-F, compare with
Fig. 1O,P). Together, these
data show that proximity to a Wnt source maintains cells in a proliferative
state and that this is achieved via transcriptional activation of Nmyc.
Proliferation and differentiation are often mutually exclusive cell states.
Are they achieved through independent regulation, or does one state actively
curtail the other? We addressed this question using E11.5
Nmyc-/- limb buds, in which Wnt3a beads could no longer
induce cell proliferation. Despite this, Wnt3a still repressed Sox9
(Fig. 6G,H) and blocked
chondrogenic differentiation (Fig.
6I,J). Moreover, Wnt3a beads also induced the formation of ectopic
Col1-positive connective tissue in absence of Nmyc
(Fig. 6K,L). Thus, the Wnt3a
source switched limb mesenchyme cells from a chondrogenic towards a soft
connective tissue fate, independently from its mitogenic effect. This
reinforces our hypothesis that Wnt signals re-specify cell fate, as opposed to
selectively expanding connective tissue precursors.
As Sox9 is essential for chondrogenesis
(Akiyama et al., 2002;
Bi et al., 1999), its
repression by Wnt signaling (Fig.
4) explains how Wnt signals inhibit chondrogenesis. This is
supported by the observation that deletion of the Wnt signal transducer
β-catenin leads to expansion of Sox9 expression in limb
mesenchyme (Hill et al.,
2005). Thus, Wnt controls proliferation and chondrogenic
differentiation through the independent transcriptional regulation of Nmyc and
Sox9.
Expansion determines differentiation
The finding that the limb ectoderm inhibits chondrogenic differentiation
led to various models wherein the size and location of the chondrogenic core
is determined by the size of the limb bud and the range of the inhibitory
signal (Kosher, 1979; Solursh,
1984; Wolpert,
1990). Several predictions can be made based on such models: (1)
chondrogenic cells will only appear where the distance to the ectoderm is
larger than the range of the inhibitory signal; and (2) increasing or reducing
the growth of the limb bud, without manipulating the range of the inhibitory
signal, will increase or reduce the size of the chondrogenic core, whereas the
thickness of the prospective soft connective tissue layer will remain
unchanged. As we have identified Wnt proteins as the ectodermal signal and
Nmyc as a critical growth mediator, we are able to test these predictions.
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Fig. 6. Wnt promotes proliferation of limb mesenchyme via Nmyc and
inhibits chondrogenesis independently via Sox9.
(A,B) BrdU staining on longitudinal sections through the
midregion of E11.5 forelimb bud of wild-type (A) and
Nmyc-/- littermate (B). (C) Proliferation in E11.5
limb bud cells infected with adenovirus expressing the indicated genes
(n=6). (D,E) Vehicle (D) or Wnt3a (E) beads implanted
in E11.5 Nmyc-/- limb buds do not affect BrdU labeling
(n=8). (F) BrdU density around vehicle or Wnt3a beads
implanted in E11.5 Nmyc+/- and Nmyc-/- limbs
(n=4). Wnt3a promotes proliferation 5-fold in
Nmyc+/- limb buds (P=0.0001, n=4), but
fails to promote proliferation in Nmyc-/- limb buds
(P=0.38, n=4). (G,H) Wnt3a beads repress
Sox9 in Nmyc-/- limb buds (H); vehicle beads (G)
have no effect (n=4). (I,J) Wnt3a inhibits cartilage
formation around the bead in Nmyc-/- limb buds (J);
vehicle beads have no effect (I). Cartilage stained red with Safranin O
(n=4). (K,L) Wnt3a beads (L), but not vehicle beads
(K), induce collagen 1 in Nmyc-/- limb buds
(n=4). Dorsal is upwards, distal rightwards (A,B). Scale bars: 100
µm. B, bead; car, cartilage.
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Fig. 7. Wnt couples expansion to differentiation. (A)
Axin2lacZ expression (blue) is visible throughout the
forelimb buds of E9.5 embryos (22 somites). (B) Sox9
expression (red) is absent in a nearby section through the same embryo as in
A. Expression of Sox9 can be seen in other areas of the embryo, such
as the neural tube. (C,D) At late E9.5 (27 somites),
Axin2lacZ is no longer active in the centre of the
forelimb (C), where Sox9 is now expressed (D). (E,F)
Sox9 immunostaining on longitudinal sections through the midregion of
stage-matched wild-type (E) and Nmyc-/- (F) E11.5
forelimbs. (G,H) Stage-matched E11.5 wild type (G) and
Prx1::Nmyc embryo (H, n=5). (I,J)
Sox9 immunostaining on longitudinal sections through the midregion of
the forelimbs of the embryos shown in G,H. Dorsal is upwards and distal is
rightwards (C-F,I,J). Nuclei are labeled in blue (B,D,E,F,I,J). Scale bars:
100 µm in A-F,I,J; 500 µm in G,H.
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At E9.5, limb buds have a radius of about 100 µm, which is approximately
the range of the Wnt signal (Fig.
5A-C). Indeed, reporter activity indicated that all cells were
responding to a Wnt signal (Fig.
7A), and absence of Sox9 expression indicated that no
chondrogenic cells were present (Fig.
7B). As the limb bud expanded to 200 µm, the center of the
developmental field escaped the range of the Wnt signal
(Fig. 7C), and we now observed
a chondrogenic population expressing Sox9 in this location
(Fig. 7D). Thus, initiation of
chondrogenesis is regulated by the size of the limb bud.
View larger version (25K):
[in this window]
[in a new window]
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Fig. 8. Wnt and FGF signals interact to coordinate growth and cell fate
specification during limb development. In the newly established limb bud
(E9.5), both Wnt and FGF proteins signal throughout the limb mesenchyme and
maintain all cells in a multipotent, proliferative state (indicated by
red/blue hatching, marked by Axin2 and Dusp6). Following limb outgrowth, cells
in the center of the limb are no longer within range of the signals. This
allows cell cycle withdrawal and expression of Sox9, leading to establishment
of the chondrogenic core (indicated in blue, marked by Sox9). In the
periphery, meanwhile, cells out of range of FGFs from the AER are still within
range of Wnts from the ectoderm (indicated by red hatching, marked by Nbl1),
which maintains the proliferative state at a lower level, and respecifies the
cells towards soft connective tissue fates. As a result of these processes, a
proximodistally extended organ forms with a multipotent, rapidly growing tip
and a chondrogenic core surrounded by soft connective tissues.
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If the size of the non-chondrogenic zone is determined by the range of the
Wnt signal, then it should retain its dimensions regardless of the size of the
developmental field. Nmyc-/- embryos develop smaller limb
buds (Charron et al., 1992;
Sawai et al., 1993;
Stanton et al., 1992), and, as
predicted, we observed that the non-chondrogenic, proliferative zone remained
similar in both its size (100 µm) and its location compared with the
wild-type limb bud (Fig. 6A,B).
But because the overall size of the Nmyc-/- limb bud was
reduced, proportionally more cells were under the influence of the ectodermal
Wnt signal and consequently the Sox9 domain was reduced
(Fig. 7E,F).
By manipulating Nmyc levels, we were also able to expand limb bud size: we
promoted mesenchymal expansion by over-expressing Nmyc under control
of the Prx1 promoter (Martin and
Olson, 2000). Prx1::Nmyc embryos had larger limb buds, confirming
the proliferative function of Nmyc
(Fig. 7G,H). As before, the
non-chondrogenic zone was unaffected, but the region of Sox9 was
considerably expanded (Fig.
7I,J). Thus, the size of the non-chondrogenic, proliferative zone
is independent of the size of the limb because it is controlled by the range
of the ectodermal Wnt signal. By contrast, any variation in growth at this
stage directly alters the size and location of the chondrogenic population.
Combined, these results support a model in which the size and location of the
chondrogenic core is determined by the size of the limb bud and the range of
the ectodermal Wnt signal. Moreover, they show that growth is a crucial
component of cell fate determination.
|
DISCUSSION
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In this study, we investigated how growth of an embryonic organ is
coordinated with the simultaneous segregation of cells into specific lineages.
We show that, during vertebrate limb development, many aspects of this process
are under control of two families of signaling proteins, Wnts and FGFs. The
apical ectodermal ridge (AER) is a source of FGF signals, whereas the limb
ectoderm produces multiple Wnts, including Wnt3 and Wnt6, which may perform
the functions outlined in this paper. One of our key findings is that the
combination of Wnt and FGF signals synergistically promotes proliferation
while maintaining the cells in an undifferentiated, multipotent state that is
pre-specified towards the chondrogenic lineage. Thus, withdrawal of the
signals results in cell cycle withdrawal and chondrogenic differentiation,
whereas continued exposure to ectodermal Wnt blocks chondrogenesis and
re-specifies the cells towards the other connective tissue lineages.
A template for a limb
Our findings support a model that explains how limb growth is coordinated
with the establishment of skeletal and soft connective tissues
(Fig. 8). In this model, both
Wnt and FGF proteins signal throughout the newly established limb bud. They
maintain all mesenchymal cells in an undifferentiated, proliferative state
(red/blue hatching; marked by Axin2 and Dusp6), leading to rapid outgrowth of
the limb bud. Once the cells in the centre of the bud are out of range of the
signals, they withdraw from the cell cycle, relieve the repression of Sox9,
and undergo chondrogenesis (blue, marked by Sox9). In the periphery meanwhile,
cells outside the influence of FGFs from the AER remain within range of Wnts
from the ectoderm (red hatching, marked by Nbl1). This Wnt signal maintains
proliferation and re-specifies the cells towards soft connective tissue fates.
Proliferation therefore occurs throughout the periphery and subridge region
(hatched areas, marked by Nmyc and Sdc1), and the limb bud continues to expand
in all dimensions. However, as Wnts and FGFs combine to promote proliferation
synergistically in the subridge region (red/blue hatched domain),
proximodistal growth dominates. As a result of these processes, a
proximodistally extended organ forms with a multipotent rapidly growing tip
and a chondrogenic core surrounded by soft connective tissues. The connective
tissues, in turn, control the pattern of differentiation of the immigrating
myoblasts.
How can this model be integrated with the existing insights into limb
patterning? Our model functions in absence of dorsoventral and anteroposterior
signaling centers. Indeed, limb development can tolerate loss of these
patterning systems, and the same basic structure then develops, consisting of
a skeletal core surrounded by soft connective tissues and muscle
(Chiang et al., 2001;
Litingtung et al., 2002;
Parr and McMahon, 1995). Our
model provides a template upon which the dorsoventral and anteroposterior
patterning mechanisms are superimposed, elaborating what would otherwise
become a simple fin-like structure.
An area of current debate is the process by which the limb bud is patterned
into a proximodistal series of segments. Neither progress zone nor early
specification models convincingly describe proximodistal patterning, and the
authors of those models have proposed the outline of a replacement
(Tabin and Wolpert, 2007). For
this, it is first postulated that commitment to differentiation and
chondrogenic condensation occurs as cells exit the domain influenced by FGF
signals from the AER. Second, it is postulated that the proximodistal
specification of a cell is based on the segment-specific genes it expresses at
the time it exits this undifferentiated zone
(Tabin and Wolpert, 2007).
Although the mechanisms that determine segment-specific gene expression are
poorly understood, it is hard not to notice the match with our model: we show
that the subridge region/undifferentiated zone is maintained by Wnts in
addition to FGF signals. The distal truncation following AER removal is
explained by the loss of the source of FGF signals coupled with the continued
production of Wnts by non-ridge ectoderm: synergistic gene regulation (e.g.
Nmyc) and polarized outgrowth stops, and the multipotent progenitors
start to differentiate. The subridge region becomes like any other peripheral
region, where cells are under the influence of Wnt signals, and form
connective tissues but no cartilage. Indeed, subridge cells no longer
contribute to skeletal structures following AER removal
(Dudley et al., 2002). We
provide the molecular foundation for further development of a proximodistal
patterning model.
Our model describes the formation of a basic, unembellished structure
somewhat resembling a paddle, without dorsoventral or anteroposterior pattern.
When and where could this basic limb, or Ur-limb, have evolved? An ectopic
source of FGF, placed underneath Wnt-expressing ectoderm
(Barrow et al., 2003;
Parr et al., 1993), induces
outgrowth, AER formation and ectopic limb formation
(Cohn et al., 1995). The
ability of FGF signals to induce ectopic outgrowths is not limited to the
paired appendages: in zebrafish, ectopic FGF signals can induce an ectopic
median fin (Abe et al., 2007).
This fin has a basic tissue arrangement similar to that of a paired appendage,
i.e. a skeletal core surrounded by connective tissue and muscle. It is thought
that the molecular mechanism of fin and limb development evolved in the
midline, before the origin of paired appendages
(Freitas et al., 2006). The
mechanism we have detailed provides a robust and adaptable molecular framework
that might underlie the development and evolution of appendages throughout the
vertebrate subphylum.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/19/3247/DC1
|
ACKNOWLEDGMENTS
|
|---|
This research was supported by the Howard Hughes Medical Institute, NIH
grant DK067834, NRSA-F32DE017499-01 (S.A.B.) and by a Human Frontiers Science
Program long term fellowship (D.tB.). We thank Calvin Kuo and Mark Lee for
help with generating adenoviral vectors. Nmyc+/- and
Axin2+/lacZ mutant mice were kindly provided by Drs Paul Knoepfler
and Robert Eisenman and Dr Walter Birchmeier.
|
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Costanti
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SUMMARY
INTRODUCTION
