Abstract
Despite a common understanding that Gli TFs are utilized to convey a Hh morphogen gradient, genetic analyses suggest craniofacial development does not completely fit this paradigm. Using the mouse model (Mus musculus), we demonstrated that rather than being driven by a Hh threshold, robust Gli3 transcriptional activity during skeletal and glossal development required interaction with the basic helix-loop-helix TF Hand2. Not only did genetic and expression data support a co-factorial relationship, but genomic analysis revealed that Gli3 and Hand2 were enriched at regulatory elements for genes essential for mandibular patterning and development. Interestingly, motif analysis at sites co-occupied by Gli3 and Hand2 uncovered mandibular-specific, low-affinity, 'divergent' Gli-binding motifs (dGBMs). Functional validation revealed these dGBMs conveyed synergistic activation of Gli targets essential for mandibular patterning and development. In summary, this work elucidates a novel, sequence-dependent mechanism for Gli transcriptional activity within the craniofacial complex that is independent of a graded Hh signal.
Highlights
The Hedgehog (Hh) signaling pathway has been studied for decades in contexts ranging from organogenesis to disease (Nusslein-Volhard and Wieschaus, 1980; Chang et al, 1994; Chiang et al, 1996; St-Jacques et al, 1999; Hebrok et al, 2000; Yao et al, 2002; Zhang et al, 2006)
Overlapping peaks containing a divergent’ glioma-associated oncogene (Gli)-binding motifs (dGBMs) were enriched near neural crest cell (NCC) cluster marker genes, when compared to all other clusters (Figure 6E). These analyses suggested that the presence of a dGBM was associated with genes differentially expressed in Gli2/3 and Hand2 conditional mutants and that it was enriched in NCCs that give rise to skeletal and glossal derivatives
Co-expression of Gli3 and Hand2 in the presence of the dGBM+E-box synthetic reporter resulted in a significant and synergistic upregulation of luciferase expression compared to either Gli3 or Hand2 alone (Figure 8E). These results indicated that (1) the low-affinity dGBM conveyed a distinct function from the canonical’ Gli-binding motif (cGBM), (2) the low-affinity dGBM+E-box produced synergistic transcriptional output in the presence of Gli3 and Hand2 and (3) synergistic activity was independent of a graded Hh signal, since the response was observed without Hh stimulation
Summary
The Hedgehog (Hh) signaling pathway has been studied for decades in contexts ranging from organogenesis to disease (Nusslein-Volhard and Wieschaus, 1980; Chang et al, 1994; Chiang et al, 1996; St-Jacques et al, 1999; Hebrok et al, 2000; Yao et al, 2002; Zhang et al, 2006). Gli is epistatic to Shh: the Shh-/-;Gli3-/- compound knockout has a polydactylous limb phenotype identical to the Gli mutant alone, indicating that the major role of Shh in the autopod is to modulate Gli3R formation (Litingtung et al, 2002; te Welscher et al, 2002) These classic genetic studies established the understanding that the formation of distinct Gli (activator) and Gli (repressor) gradients are necessary for converting the Hh signal transduction cascade into downstream gene expression responses within the vertebrate NT and limb. Our findings suggest that context-dependent optimization of Gli- binding site occupancy in the presence of Hand is critical for modulating tissue-specific transcriptional output within a tissue that lacks an obvious Shh morphogen gradient These findings define how craniofacial prominences can serve as distinct developmental fields that interpret Hh signals in a manner unique to other organ systems
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