A regulatory network of two galectins mediates the earliest steps of avian limb skeletal morphogenesis

Here we provide evidence that galectins, β-galactoside-binding lectins with β-sandwich folding, play fundamental roles in these processes. We show that among the five chicken galectin (CG) genes, two, CG-1A, and CG-8, are markedly elevated in expression at prospective sites of condensation in vitro and in vivo, with their protein products appearing earlier in development than any previously described marker. The two molecules enhance one another's gene expression but have opposite effects on condensation formation and cartilage development in vivo and in vitro: CG-1A, a non-covalent homodimer, promotes this process, while the tandem-repeat-type CG-8 antagonizes it. Correspondingly, knockdown of CG-1A inhibits the formation of skeletal elements while knockdown of CG-8 enhances it. The apparent paradox of mutual activation at the gene expression level coupled with antagonistic roles in skeletogenesis is resolved by analysis of the direct effect of the proteins on precartilage cells. Specifically, CG-1A causes their aggregation, whereas CG-8, which has no adhesive function of its own, blocks this effect. The developmental appearance and regulation of the unknown cell surface moieties ("ligands") to which CG-1A and CG-8 bind were indicative of specific cognate- and cross-regulatory interactions.Our findings indicate that CG-1A and CG-8 constitute a multiscale network that is a major mediator, earlier-acting than any previously described, of the formation and patterning of precartilage mesenchymal condensations in the developing limb. This network functions autonomously of limb bud signaling centers or other limb bud positional cues.The formation of the vertebrate limb skeleton has become a paradigm of developmental biology due to its suitability for the study of processes such as tissue induction, cell differentiation, and spatial patterning of differentiated cell types [1]. A key cellular event in the early developing limb is the formation of condensations - high-densi