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 (), and inhibited chondrogenesis in the mesenchymal cells around it (). In the presence of the Wnt antagonist Fz8CRD (Hsieh et al., 1999
), Wnt reporter activity was markedly reduced () and chondrogenesis occurred (). These data indicate that limb mesenchyme responds to a Wnt signal from the ectoderm and that this signal inhibits chondrogenesis.
Limb ectoderm inhibits chondrogenesis and promotes proliferation via Wnt signals
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 () and chondrogenesis in these cells was inhibited (). 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 () and the protein alone was sufficient to block chondrogenic differentiation in cells around the Wnt source (). 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 (). Moreover, this effect depends on Wnt signals as it was abolished by the Wnt antagonist Fz8CRD (). 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 (), a strong increase in BrdU labeling was observed around Wnt3a beads (). Combined, our data show that the limb ectoderm, by secreting Wnts, not only inhibits chondrogenic differentiation but also promotes proliferation in the underlying mesenchyme.
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
; ), 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 (). Over the course of 8 days, the cells upregulated expression of collagen 1, tenascin C, decorin, Dermo1 and Bmp3 (), whereas expression of scleraxis or the (pre)osteoblast marker osteopontin was not detected (). 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
Wnt3a protein re-specifies chondrogenic cells towards soft connective tissues
Connective tissue markers and their expression in response to Wnt3a in limb mesenchyme cells
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 (). But as Dermo1 and Bmp3 were not induced (), 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 (). Continuous exposure to Wnt3a protein, which does not promote formation of muscle connective tissue, led to a small increase in the number of myotubules (), 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 ( and data not shown). By contrast, Wnt3a beads were never in contact with cartilage (, and see ) but disrupted the pattern of cartilage differentiation and that of muscle and connective tissue (). 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 (). Combined staining for muscle fibers and pro-collagen 1-positive connective tissue demonstrated that this was ectopic connective tissue (). 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.
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 (), whereas the combination of Fgf8 with Wnt3a inhibited chondrogenesis altogether (). 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 ().
Wnt and FGF proteins act in synergy to promote proliferation and maintain the undifferentiated state
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 (). By contrast, when derived from cells expanded in presence of Wnt3a and Fgf8, the micromasses differentiated into cartilage, similar to freshly isolated limb mesenchyme (). Moreover, Wnt3a was still able to inhibit this chondrogenesis (), 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 (). However, Fgf8 enhanced the proliferative effect of Wnt3a (). 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 (). Alcian Blue staining revealed that the extra tissue was largely of a chondrogenic nature (), 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.
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 (): 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 syndecan 1/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
Regulation of target genes by Wnt3a and Fgf8 in chick limb mesenchyme
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 (), 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
, another gene induced by Wnt alone (), is similarly expressed (Jukkola et al., 2004
Expression patterns of Wnt and FGF target genes correlate with cell behaviors in mouse limb buds
Our data show that Wnt stimulates proliferation () and BrdU labeling indeed occurred predominantly in the Wnt-responsive region of the limb (). Wnt3a and Fgf8 synergize in promoting proliferation in culture () and we found that proliferation is highest in the subridge region where Wnt and FGF signals overlap () (Pascoal et al., 2007
). A similar pattern of expression was displayed by Nmyc
() (Sawai et al., 1993
; Solursh et al., 1990
), in accordance with their synergistic induction by Wnt and FGF signals ().
Wnt signals inhibit cartilage differentiation (). 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 (). Nbl1
is induced by Wnt3a, and this induction is antagonized by Fgf8 in vitro (); 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 (). In contrast to Nbl1
is expressed underneath the AER, although slightly weaker ventrally, suggesting that this antagonism is not strong enough to overcome the inducing signal (). 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 (). 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 () and in the absence of Nmyc
, cell division in this zone was dramatically reduced (). 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 (, one-way ANOVA, P
Wnt promotes proliferation of limb mesenchyme via Nmyc and inhibits chondrogenesis independently via Sox9
To determine whether Nmyc
was required for Wnt-induced proliferation, we impaired Nmyc function by overexpressing a dominant-negative form of the gene, NmycΔMB2
(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 NmycΔMB2
-overexpressing cells (P
=0.1809) (). 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
=0.0820) (). 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 (, compare with ). 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 () and blocked chondrogenic differentiation (). Moreover, Wnt3a beads also induced the formation of ectopic Col1-positive connective tissue in absence of Nmyc (). 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 () 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.
At E9.5, limb buds have a radius of about 100 µm, which is approximately the range of the Wnt signal (). Indeed, reporter activity indicated that all cells were responding to a Wnt signal (), and absence of Sox9 expression indicated that no chondrogenic cells were present (). As the limb bud expanded to ~200 µm, the center of the developmental field escaped the range of the Wnt signal (), and we now observed a chondrogenic population expressing Sox9 in this location (). Thus, initiation of chondrogenesis is regulated by the size of the limb bud.
Wnt couples expansion to differentiation
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 (). 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 ().
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
(). As before, the non-chondrogenic zone was unaffected, but the region of Sox9
was considerably expanded (). 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.