A SHH-independent regulation of Gli3 is a significant determinant of anteroposterior patterning of the limb bud.

The family of GLI proteins (GLI1-3) comprises the intracellular mediators of the hedgehog pathway, which regulates a myriad of developmental processes, one of which is limb development. Whereas GLI1 and GLI2 seem to be dispensable during limb development, GLI3 is especially crucial since all GLI3-associated human congenital diseases comprise limb malformations. Furthermore, Gli3(-/-) mouse embryos exhibit pronounced polydactyly in conjunction with a loss of digit identities. Here we examined how the quantity of GLI3 contributes to its function by using different Gli3 mutants in order to vary overall GLI3 levels. In addition, we made use of the Gli3(Delta699) allele, which encodes a C-terminally truncated version of GLI3, thus mimicking the processed GLI3 isoform (GLI3R). The Gli3(Delta699) mutant made it feasible to analyze isoform-specific contributions of GLI3 within the context of anteroposterior patterning of the limb bud. We revealed a so far unappreciated variation in the quantitative demand for GLI3 within different phases and aspects of distal limb formation. In addition, our analyses provide evidence that unprocessed full-length GLI3 is dispensable for anteroposterior patterning of the limb bud. Instead, digit identities are most likely defined by GLI3 repressor activity alone. Furthermore, we present evidence that the anteroposterior grading of GLI3 activity by the action of SHH is supported by a prototype patterning, which regulates Gli3 independently from SHH.