Gli3 (Xt) and formin (Id) participate in the positioning of the polarisingregion and control of posterior limb-bud identity

During initiation of limb-bud outgrowth in vertebrate embryos, the polarisi ng region (limb-bud organizer) is established upon activation of the Sonic Hedgehog (SHH) signaling molecule at the posterior limb-bud margin. Another hallmark of establishing anteroposterior limb-bud identities is the coline ar activation of HoxD genes located at the 5' end of the cluster (5'HoxD ge nes). The unique and shared functions of Gli3 and formin in these determina tive events were genetically analyzed using single and double homozygous Ex tra-toes (Xt; disrupting Gli3) and limb deformity (ld; disrupting formin) m ouse embryos. Analysis of the limb skeletal phenotypes reveals genetic inte raction of the two genes. In addition to loss of digit identity and varying degrees of polydactyly, proximal skeletal elements are severely shortened in Xt;ld double homozygous limbs. The underlying molecular defects affect b oth establishment of the polarising region and posterior limb-bud identity. In particular, the synergism between Gli3- and forinin-mediated mesenchyme -AER interactions positions the SHH signaling center at the posterior limb- bud margin. The present study shows that establishment and positioning of t he polarising region is regulated both by restriction of Shh through Gli3 a nd its positive feedback regulation through formin, Concurrently, Gli3 func tions independently of formin during initial posterior nesting of 5'HoxD do mains, whereas their subsequent distal restriction and anterior expansion d epends on genetic interaction of Gli3 and formin.