The Effection Of The Ratio Of Gli2FL And Gli2Rep On The Activity Of Hedgehog Signaling Pathway And Mouse Development

Background and PurposeIn mice, Hh signaling is mediated by three Gli transcription factors, Gli1, Gli2, and Gli3. The functions of the three Gli proteins overlap but also are distinct. This is mostly determined by the intrinsic molecular nature of the proteins. In the absence of Hh signaling, the majority of the full-length Gli3 protein, Gli3FL, is proteolytically processed to the Gli3 transcriptional repressor, Gli3Rep. Gli3 processing is dependent on the phosphorylation of multiple serine and threonine residues at its C-terminus by protein kinase A (PKA) and subsequently by casein kinase 1 (CK1) and glycogen synthase kinase 3 (GSK3). Once phosphorylated, Gli3FL is bound and ubiquitinated by the SCFβTrCP ubiquitin E3 ligase and consequently processed by the proteasome in a site-specific manner to generate the Gli3 repressor. Hh signaling inhibits Gli3 processing and activates the Gli3FL, which leads to upregulation of the expression of Gli target genes. However, even activated, Gli3FL exhibits a weak activator function in vivo. Therefore, Gli3 overall plays an inhibitory role in Hh signaling.Gli2 is thought to be a primary transcriptional activator that mediates Hh signaling. Although, like that of Gli3FL, the mouse Gli2FL is also processed to generate a repressor, but the extent of its processing is much lower than that of Gli3. The differential processing between Gli2 and Gli3 is determined by the processing determinant domain (PDD), a region of 197-amino acid residues between the zinc-finger DNA binding domain and the first PKA site of the proteins. Gli2 acts only as an activator, however, genetic studies of somite development of Gli2 mutants in a Gli3 null background have uncovered a weak repressor activity of Gli2, which is consistent with the presence of low level of Gli2 repressor. Thus, it remains controversial whether Gli2 exhibits a repressive function in Hh signaling, and this needs to be clarified. In order to determine the role of Gli2 repressor in Hh pathway and mouse development, we replaced Gli2PDD with Gli3PDD and generated a Gli2 mutant allele, Gli23PDD to detect the Hh pathway activity in vivo, which helps us to understand the importance of the proper balance between the Gli2FL and repressor in mouse development..Methods1. ES cells culture; Generation of the Gli23PDD mutant knock-in allele; CaCl2+HBS transfection; Western Blotting2. Dissection of embryos; Immunohistochemistry of frozen section3. Generation of mouse embryonic fibroblasts (MEFs); in situ hybridization; RT-PCR4. Reporter assay; Lipfetamine transfection; ImmunoblottingResults1. Differential Gli2 and Gli3 processing is conserved between the mouse and the zebrafish2. A change in the balance between the Gli2FL activator and its repressor does not alter the neural tube patterning but results in a slight reduction of mouse body weight3. A Change in the balance between the Gli2FL activator and its repressor reduces the Hh pathway activity4. The transcription activity of Gli2-3PDD reduces and its processing is regulated by Hh signalingConclusionIn this study, we conclude that differential Gli2 and Gli3 processing is conserved between the mouse and the zebrafish, and it is essential for the distinct function of Gli2 and Gli3. To verify the observation in vivo, we genetically engineered a Gli23PDD mutant allele in which the last third and second coding exons of the mouse Gli2 gene that encode 585-751 amino acid residues were replaced by corresponding Gli3 cDNA sequence. The levels of Gli2 repressor in Gli23PDD mutant embryos was more than two times of that of wild type Gli2 repressor while the Gli23PDD full length protein was only slightly lower than wt Gli2FL. In addition, the Gli2 repressor resulted from Gli23PDD processing was slightly smaller than that of wt Gli2 repressor, indicating that the processing site of the Gli23PDD protein has shifted compared to that of wt Gli2FL. The mutant mice were alive and fertile without any noticeable phenotypes. However, the homozygous mutant mice on average were slightly smaller than wt or heterozygotes siblings at 2.5,3.5, and 7 weeks of age that were examined. The changes in the ratio of Gli2FL to Gli2Rep in Gli23PDD mutant does not lead to a detectable defect in neural tube patterning. However, the levels of Glil and Ptchl expression in both spinal cord and limb bud of Gli23PDD mutant embryos were consistently found to be slightly lower than those in wt embryos but higher than those in Gli2zki null mutant. Thus, the change in the ratio of Gli2FL to Gli2Rep in Gli23PDD mutant indeed reduces the activity of Gli2 transcriptional and hedgehog pathway. Besides, Gli-dependent luciferase reporter showed that Gli23PDD transcription activity was higher than wt Gli3 and lower than Gli2. The reason why net Gli2 transcriptional activity was reduced was most likely because of the consequence of the change in the ratio of Gli2Rep to the full-length Gli23PDD, but not the full-length Gli23PDD, since the mutant protein can effectively respond to Hh signaling. These results suggest that maintaining a proper balance between the Gli2FL activator and the Gli2Rep is important for Hh signaling.