The Role Of Gli3Repressor In Limb Patterning And The Role Of Talpid~3Gene In Hedgehog Signal Transduction And Primary Cilia Formation

The vertebrate limb is a powerful model for investigating pattern formation in embryo developement. Although limb shape varies in different species, recent studies demonstrate that a significant overlap exists in the signals used to pattern limbs. In developing embryos, limb comes from the somatic mesoderm and ectoderm and is controlled by three main axes:the anteroposterior (AP), dorsoventral (DV) and the proximodistal (PD).The anterioposterior vertebrate limb patterning is thought to be controlled by counteraction between Sonic Hedgehog (Shh) and the Gli3transcriptional repressor. However, mouse limbs expressing only a Gli3repressor do not exhibit phenotypes that resemble those of Shh mutant. These findings are inconsistent. Therefore, it remains debatable whether Shh patterns the anterioposterior limb through only the inhibition of the Gli3repressor function.In the present study, we created a conditional Gli3mutant allele that expresses only the Gli3repressor in the presence of Cre recombinase. Using this mutant, we showed that the phenotypes of mouse limbs expressing only the Gli3repressor exhibited no or single digit, which resemble those of Shh mutant limbs. Consistent with the limb phenotypes, the expression of the genes that are normally dependent on Shh signaling are lost in both this Gli3repressor mutant and Shh mutant. The inhibition of the gene expression by the Gli3repressor is independent of the Shh expression. Thus, our study clarifies the current controversy and provides an important piece of genetic evidence to support the hypothesis that Shh patterns the anterioposterior limb by solely inhibiting the Gli3repressor function.Recent findings indicate that mammalian Shh signal transduction occurs within primary cilia, but the biological mechanisms of Shh signaling and ciliogenesis have not been fully elucidated. The chicken talpid3gene is found to encode a novel protein essential for intraflagellar transport, ciliogenesis and Shh signal transduction. Here we generate a Talpid3mutant mouse and found the primary cilia was lost in these mustant cells. We found the Talpid3mutant cells have multiple centrosomes and the localization of centrosomal protein120(Cep120) was also changed. Based on the pull-down and mass spectrometry results, we further show the interation between Talpid3and PKA through the coiled-coil domain of Talpid3. We propose that Talpid3serves as an anchor protein, which recruits the Cep120to localize in the daughter centriole and assemble the basal body for ciliogenesis. As an anchor protein, Talpid3also interacts with PKA, allowing PKA to release the catalytic domain and phosphorylate the down stream transcriptional factors of Shh signal.