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(Delta 699) allele, which encodes a C-terminally truncated version of GLI3, thus mimicking the processed GLI3 isoform (GLI3R). The Gli3(Delta 699) 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. (C) 2009 Elsevier Inc. All rights reserved.

Organization of the vertebrate inner ear is mainly dependent on localized signals from surrounding tissues. Previous studies demonstrated that sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of ventral (auditory) and dorsal (vestibular) inner ear structures, yet it was not clear how this signaling activity is propagated. To elucidate the molecular mechanisms by which Shh regulates inner ear development, we examined embryos with various combinations of mutant alleles for Shh, Gli2 and Gli3. Our study shows that Gli3 repressor (R) is required for patterning dorsal inner ear structures, whereas Gli activator (A) proteins are essential for ventral inner ear structures. A proper balance of Gli3R and Gli2/3A is required along the length of the dorsoventral axis of the inner ear to mediate graded levels of Shh signaling, emanating from ventral midline tissues. Formation of the ventral-most otic region, the distal cochlear duct, requires robust Gli2/3A function. By contrast, the formation of the proximal cochlear duct and saccule, which requires less Shh signaling, is achieved by antagonizing Gli3R. The dorsal vestibular region requires the least amount of Shh signaling in order to generate the correct dose of Gli3R required for the development of this otic region. Taken together, our data suggest that reciprocal gradients of GliA and GliR mediate the responses to Shh signaling along the dorsoventral axis of the inner ear.

Mutations in GLI3 manifest in several distinct clinical phenotypes including Greig cephalopolysyndactyly syndrome and Pallister-Hall syndrome (PHS). GLI3 belongs to the GLI family of transcription factors that mediates extracellular Sonic hedgehog (SHH) signals. In the absence of SHH signals, GLI3 is processed to form a transcriptional repressor termed GLI3R. During early limb development, the regulation of GLI3 processing by SHH is decisive in determining the correct number and identity of digits. Analyses of mouse embryos have produced evidence that elevated levels of GLI3R reduce the number of developing digits. Remarkably, PHS causative mutations are predicted to produce a truncated protein similar to the endogenous GLI3R. Nevertheless, polydactyly is frequently observed in PHS patients and it even represents a criterion for the clinical diagnosis of PHS. In order to detect the underlying cause of this obvious discrepancy, we made use of the Gli3(Delta699) mouse mutant, which represents the mouse model of PHS. We show that the mutant murine allele gives rise to a truncated version of GLI3 that mimicks both the processed GLI3R isoform and the proposed pathogenic GLI3(PHS) protein. We analyzed how the mutant GLI3 protein interferes with the anteroposterior patterning of early limb development, whereas processes that are associated with the outgrowth of the limb bud remain remarkably unimpaired. The presented findings help to understand the previously enigmatic emergence of Pallister-Hall associated polydactyly and thus add to the understanding of the pathogenic mode of the action of GLI3(PHS).