Our results show that the limb ectoderm is a negative regulator of the earliest stages of chondrogenesis in developing chick limbs upstream of Sox9. We furthermore demonstrate that Wnt6, which is, in the chick, expressed in both dorsal and ventral limb ectoderm [24, 25], inhibits chondrogenesis upstream of Sox9 expression, arguing for an early inhibiting activity of ectodermal Wnt6 prior to chondrocyte differentiation. At present Wnt6 is the only Wnt known to be expressed throughout the avian limb ectoderm  (Fig. 4). Our data do not exclude that other Wnts, like Wnt7a expressed in the dorsal ectoderm  or other yet unidentifed ectodermal Wnts, also participate in this function. This redundancy is very probable in the mouse, where six Wnt genes (Wnt3, Wnt4, Wnt6, Wnt7b, Wnt9b, Wnt10a) have been described to be coexpressed in the limb ectoderm , and several Wnts, including Wnt3a, have been shown to be inhibitors of chondrogenesis [23, 28].
A regulatory role of the ectoderm during differentiation of the limb mesenchyme has already been proposed by Blechschmidt in 1963 . Classical studies have experimentally confimed a role of the limb ectoderm during cartilage differentiation [30–32], whereas a regulatory role of the muscle anlagen on skeletogenesis can be excluded as muscle-less limbs form normal skeleton . In vitro investigations have suggested that the limb ectoderm produces a diffusible factor which inhibits chondrogenic differentiation in the underlying mesenchyme. Limb ectoderm from stage 23/24 wing buds has been shown to inhibit cartilage differentiation of cultured limb mesenchyme cells even without direct contact but acting over some distance, thus arguing for a secreted mechanism of inhibition [9, 10]. This is in line with the long range inhibitory activity of ectodermal Wnt6 in the limb suggested by our experiments.
The sequential steps of chondrogenesis are characterized by the expression of typical marker genes. Initially, the uniform limb mesenchyme aggregates in the central core of the limb mesenchyme to form precartilaginous mesenchymal condensations, which roughly presage the future skeletal elements. These prechondrocytic mesenchymal cells produce high levels of hyaluronic acid and cell adhesion proteins like N-CAM and N-Cadherin. During chondrogenic differentiation, the nascent chondrocytes express the nuclear transcription factor Sox9, which is required for the expression of the cartilage-specific marker gene ColIIA1, and start to deposit the cartilage matrix including Collagens II, IX, XI, and aggrecan. Subsequently, the balance between chondrocyte differentiation and proliferation is regulated by interaction of members of the BMP, FGF and Ihh pathways, which eventually leads to chondrocyte hypertrophy and osteogenesis (reviewed in ).
Akiyama et al.  have shown that Sox9 is required during at least two steps of cartilage formation, first during mesenchymal condensation and second, as an inducer of the related factors Sox5 and Sox6, during overt chondrocyte differentiation. In line with this, early inactivation of Sox9 leads to a loss of cartilage and bone . Canonical Wnt signaling, which is known to inhibit chondrogenesis [18, 19], is antagonizing Sox9 activity at posttranslational level . Accordingly, forced expression of beta-catenin, a member of the canoncial Wnt pathway, in prechondrogenic cells leads to shortened or missing skeletal elements , a skeletal phenotype similar to the skeletal defects described here after Wnt6 overexpression. Moreover, stabilization of beta-catenin in mouse limbs leads to repression of Sox9, whereas deletion of beta-catenin results in an expansion of the Sox9 expression domain in the limb bud mesenchyme . This argues for the hypothesis that the inhibitory effect of Wnt6 on chondrogenesis observed in this report could be transduced by the canoncial Wnt pathway, as has been described for the epithelializing activity of Wnt6 in the somites . However, as Wnt6 has also been shown to act via non-canonical signaling , further studies on the downstream events leading to chondrogenic inhibition will be required to substantiate this hypothesis.
As we found that Wnt signaling not only inhibits Sox9 expression, but also expression of the early Sox9 target gene ColIIA, we argue that Wnt6 signaling negatively regulates chondrogenesis at a very early step, likely during mesenchymal condensation. This is in agreement with a recent study identifying Sox9 as a target of Wnt3a -mediated inhibition of cartilage formation .
Interestingly, also overexpression of Sox9 has been observed to result in shortened skeletal elements, likely due to inhibition of cell proliferation and differentiation . This illustrates that the chondrogenesis-promoting activity of Sox9 is finely balanced. As activated beta-catenin and Sox9 are interacting in a negative feedback loop , correct Sox9 activity depends on an appropriate level of canonical Wnt signaling.
In accordance with this, we report here that the severity of limb skeletal defects is correlated with the amount of Wnt6 expressing cells implanted. This finding, and data from earlier work from our laboratory , argue for a fine-tuned, dose-dependent regulatory activity of ectodermal Wnt signaling on the development of both, muscle and cartilage. Our results support a model of centripetal patterning of the limb by ectodermal Wnt signaling: Within the peripheral limb mesenchyme, which is close to the ectodermal source of Wnt6, immigrated muscle precursor cells originating from the somites receive high doses of Wnt ligand, and differentiate into muscle , whereas the chondrogenic pathway is inhibited. The deeper, centrally located autochthonous mesenchyme, which is farther away from the ectodermal source of Wnt6, receives low doses of Wnt ligand, thus allowing for Sox9 expression and cartilage formation in the central limb. Our data are in line with a previously proposed model combining Wnt and FGF signaling in proximodistal and centripetal limb patterning . In extension to this model, our results indicate that ectodermal Wnt signaling inhibits chondrogenesis upstream of Sox9, and provide in vivo data identifying Wnt6 as a candidate inhibitor. Recent results indicate that in addition to the Wnt gradient, local expression of Wnt-inhibitors like Wif-1 at the cartilage-mesenchyme-interface impedes Wnt-mediated inhibition of cartilage formation in the skeletogenic regions , possibly thus sharpening the circumference of the forming skeletal elements. Using Axin2lacZ/+ mouse mesenchyme as reporter, Nusse and coworkers measured the reach of ectodermal Wnt signaling leading to a mesenchymal intracellular response to be 100 μm, which is in line with a Wnt-dependent centripetal patterning activity originating in the ectoderm . Thus our data support the hypothesis that ectodermal Wnt signaling acts on the limb mesenchyme in a centripetal dosage gradient which is involved in specifying mesenchymal differentiation into peripheral muscle and central cartilage.