Wnt regulation of limb mesenchymal chondrogenesis is accompanied by altered N-cadherin-related functions

It has been suggested that Wnt proteins play essential roles in the regulation of limb development and morphogenesis. We have analyzed the effects of RCAS retroviral-mediated misexpression of Wnt-5a and Wnt-7a, two Wnt genes expressed during normal limb development, on chondrogenesis in high-density micromass cultures of chick limb bud mesenchymal cells. Wnt-7a misexpression inhibited chondrogenesis in vitro, whereas Wnt-5a did not. Analysis of cellular condensation and precartilage aggregate formation revealed that the inhibitory effect of Wnt-7a is unlikely to act at the cellular condensation step but, instead, influences the actual chondrogenic differentiation process. During early condensation, N-cadherin expression within the cellular aggregates in Wnt-7a cultures was slightly delayed, whereas Wnt-5a cultures appeared similar to control cultures. With the initiation of differentiation, Wnt-5a and control cultures showed down-regulation of N-cadherin and up-regulation of collagen type II expression, whereas Wnt-7a cultures showed persistent, high levels of N-cadherin expression both at the mRNA and protein levels, up to 72 h, and nearly no collagen type 11 expression. Wnt-7a misexpression was also accompanied by an initial increase in the serine/threonine phosphorylation of beta-catenin and N-cadherin, events known to affect N-cadherin turnover and stabilization of beta-catenin/N-cadherin cell adhesion complex, during chondrogenic differentiation. Writ regulation of limb mesenchymal chondrogenesis is thus likely to involve modulation of N-cadherin expression and function. In addition, Wnt-7a misexpression stimulates the proliferation of limb mesenchymal cells in vitro, possibly contributing to its chondro-inhibitory effect. Finally, the differences between Wnt-5a and Wnt-7a effects suggest functional diversity within the Wnt family.