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

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 Gli3Δ699 allele, which encodes a C-terminally truncated version of GLI3, thus mimicking the processed GLI3 isoform (GLI3R). The Gli3Δ699 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.