Wnt11 negatively regulates early chondrogenesis in a cell-type-dependent manner in embryonic mouse mesenchymal cells

Wnt signaling is a key regulator of skeletal development. The ligand-specific roles of Wnt signaling during early chondrogenesis remain incompletely understood. WNT11, a non-canonical Wnt ligand, has established functions in embryonic morphogenesis, but its contribution to primary chondrogenesis and whether this differs across distinct mesenchymal lineages is unknown. Using embryonic mouse mandibular processes and limb bud micromass cultures, we investigated Wnt11 function through siRNA-mediated knockdown, recombinant WNT11 treatment, and pharmacological Wnt inhibition with the IWP-2. Both cell types underwent spontaneous chondrogenic differentiation, with limb bud cells showing higher baseline output. IWP-2 inhibitor enhanced chondrogenesis in both cell types, more strongly in mandibular process cells, and rapidly suppressed JNK phosphorylation while accelerating SOX9 activation. Knockdown of Wnt11 increased cartilage nodule formation and upregulated chondrogenic markers, while exogenous recombinant WNT11 treatment inhibited differentiation in a cell-specific manner in mandibular process cultures. Rescue experiments indicated that recombinant WNT11 could significantly rescue the pro-chondrogenic effects of IWP-2 inhibition in mandibular processes but not limb bud cultures. These data identify Wnt11 as a cell-type-dependent negative regulator of early chondrogenesis. This is the first direct comparison of Wnt11 function in craniofacial and appendicular embryonic mesenchymal cells, showing lineage-dependent differences with possible implications for craniofacial skeletal development.