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. Author manuscript; available in PMC: 2012 Apr 1.
Published in final edited form as: Dev Dyn. 2011 Feb 18;240(4):766–774. doi: 10.1002/dvdy.22578

Increased proteolytic processing of full-length Gli2 transcription factor reduces the Hedgehog pathway activity in vivo

Juan Li 1,2, Chengbing Wang 2, Yong Pan 2,4, Zengliang Bai 1, Baolin Wang 2,3,*
PMCID: PMC3071291  NIHMSID: NIHMS270239  PMID: 21337666

Abstract

The proteolytic processing of Gli2 and Gli3 full-length transcription factors into repressors is a key step of the regulation in Hedgehog (Hh) signaling. The differential Gli2 and Gli3 processing is controlled by the processing determinant domain or PDD, but its significance is not clear. We generated a Gli2 mutant allele, Gli23PDD, in which the Gli3PDD substitutes for the Gli2PDD. As expected, Gli23PDD is processed more efficiently and at the different position as compared to Gli2, indicating that PDD also determines the extent and site of Gli2 and Gli3 processing in vivo. The increase in levels of the Gli2 repressor in Gli23PDD mutant reduces the Hh pathway activity. Gli23PDD processing is still regulated by Hh signaling. These results indicate that the proper balance between the Gli2 full-length activator and repressor is essential for Hh signaling.

Keywords: Gli2, Hedgehog, proteolytic processing

Introduction

The Hedgehog (Hh) family of secreted signaling molecules plays fundamental roles in the patterning of many embryonic structures of animals ranging from flies to humans (Huangfu and Anderson, 2006; Jiang and Hui, 2008). Hh signals by binding to and antagonizing the twelve-pass membrane protein Patched (Ptch). This alleviates the suppression of the seven-transmembrane protein Smoothened (Smo), which in turn initiates an intracellular signaling cascade.

In 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 (Wang et al., 2000). 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 (Tempe et al., 2006; Wang and Li, 2006). Hh signaling inhibits Gli3 processing and activates the Gli3FL, which leads to upregulation of the expression of Gli target genes (Wang et al., 2000; Huangfu and Anderson, 2005). However, even activated, Gli3FL exhibits a weak activator function in vivo (Wang et al., 2007). Therefore, Gli3 overall plays an inhibitory role in Hh signaling (Vortkamp et al., 1991; Hui and Joyner, 1993).

In contrast to Gli3, Gli1 is a potent transcriptional activator, and its transcription is directly regulated by Hh signaling (Hynes et al., 1997; Dai et al., 1999; Bai and Joyner, 2001). This is mostly because the protein lacks a repressor domain and the proteolytic processing (Dai et al., 1999; Sasaki et al., 1999; Kaesler et al., 2000). Nevertheless, Gli1 is not essential for initial Shh signal transduction in mice as its gene is dispensable (Park et al., 2000; Bai et al., 2002). Thus, Gli1 appears to enforce the expression of Hh target genes upon their initial activation by Hh signaling in the mouse.

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, the extent of its processing is less than that of Gli3 (Pan et al., 2006). 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 (Pan and Wang, 2007). Genetic analysis of Gli2 null mutant mice has led to different conclusions as to whether Gli2 exhibits any transcriptional repressive activity in the Hh signaling. On one hand, the observation that expression of Gli1 from the Gli2 locus in Gli1 knock-in mice can rescue Gli2 mutant phenotypes appears to support the notion that Gli2 acts only as an activator (Bai and Joyner, 2001). On the other hand, genetic studies of somite development of Gli2 mutants in a Gli3 null background have uncovered a weak repressor activity of Gli2 (Buttitta et al., 2003; McDermott et al., 2005), which is consistent with the presence of low level of Gli2 repressor (Pan et al., 2006). Thus, it remains controversial whether Gli2 exhibits a repressive function in Hh signaling, and this needs to be clarified.

To investigate the significance of the extent of Gli2 processing, we took a genetic approach to alter the ratio of the Gli2FL activator to its repressor in the mouse. Characterization of the phenotypes of the mutant mice showed that a decrease in the ratio of the Gli2FL activator to its repressor reduces the Hh pathway activity. Therefore, our data support the notion that maintaining a proper ratio of the Gli2 full-length form to the Gli2 repressor plays a role in Hh signaling.

Results

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 weight

We have previously shown that the differential Gli2 and Gli3 processing in cultured cells is determined by a processing determinant domain (PDD), a region of nearly 200 amino acid residues located between the zinc finger DNA-binding domain and the first PKA site in the C-termini of Gli2 and Gli3 proteins (Pan and Wang, 2007). To verify this 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 (Fig. 1A–C, and material and methods). Immunoblotting analysis of protein lysates made from E10.5 embryos showed that levels of the Gli2 repressor in Gli23PDD mutant embryos was more than two times of wild type (wt) Gli2 repressor while the Gli23PDD full-length protein was lower than wt Gli2FL. As a consequence, the ratio of Gli2FL to Gli2Rep changed from approximately 5 in wt to 1.5 in the mutant. In addition, the Gli2 repressor resulted from Gli23PDD processing was slightly smaller than that of wt Gli2 repressor (Fig. 1D, compare lane 1 to lane 2), indicating that the processing site of the Gli23PDD protein has shifted compared to that of wt Gli2FL. This is in agreement with our previous finding that replacing Gli2 PDD with Gli3 PDD in the Gli2 protein shifted the position of Gli2 processing in cultured cells (Pan and Wang, 2007). Together, these results show that the PDD indeed determines the extent and position of Gli2 and Gli3 processing in vivo.

Figure 1. Gli23PDD is more efficiently processed than wt Gli2 in vivo.

Figure 1

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(A) The gene targeting strategy used to create Gli23PDD allele and ES cell screening. Open boxes indicate Gli2 exons, and the filled box is referred to Gli3 PDD sequence. Asterisks above the last exon stand for six PKA sites. The Gli23PDD targeting construct was created by replacing a part of the last third and entire last second coding exons (585–751 aa) of the Gli2 gene with its corresponding Gli3 cDNA sequence (648–814 aa). The neomycin (neo) gene flanked by loxP sites (triangles) and diphtheria toxin A (DTA) gene were used as positive and negative selection markers, respectively. Thick lines represent probes used for Southern blot analysis. The relevant restriction sites are: B, BamHI; Bg, BglII; Bs, BstZ17I; E, EcoRI; H3, HindIII; and Sp, SpeI. (B) Southern blot analysis of representative ES cell (ESC) clones using the 5'- and 3'-probes shown in A following EcoRI or HindIII digestion, respectively. Arrows indicate the expected sizes of the digested DNA fragments for the mutant and wt Gli2 alleles. (C) Diagram showing the expected full-length wt Gli2 and Gli23PDD proteins. (D) Immunoblots showing that the Gli23PDD protein is more efficiently processed than wt Gli2. Protein lysates prepared from E10.5 wt and Gli23PDD/3PDD mouse embryos were incubated with biotinylated normal (Gli-oligo) or mutated (ctrl-oligo) Gli-binding oligonucleotides and precipitated with Streptavidin-conjugated magnetic beads and then immunoblotted with a Gli2 antibody. Arrows indicate the full-length Gli2 protein, Gli2FL, and the processed Gli2 repressor, Gli2Rep. Shown in the graph are the relative levels of Gli2 proteins. Note that the Gli2Rep from Gli23PDD migrated faster than that of wt Gli2 and that its level was also higher.

To determine the significance of an increase in the levels of Gli2 repressor in Gli23PDD mutant, the gross phenotypes of mice homozygous for Gli23PDD were characterized. The mutant mice were alive and fertile without any noticeable phenotypes. However, the homozygous mutant mice on average were slightly smaller than wt or heterozygous siblings at 2.5, 3.5, and 7 weeks of age that were examined, although the difference was not statistically significant as the P values of the Student’s t-test were close to or greater than 0.05 (Fig. 2). These data suggest that Gli23PDD mutation slightly affected mouse growth.

Figure 2. Mice homozygous for Gli23PDD are slightly but not significantly smaller than wild type or Gli23PDD heterozygotes.

Figure 2

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The graph shows the average body weight of 10 animals from each sex and genotype at 2.5, 3.5, and 7 weeks of age and standard deviations. P values for the Student’s t-test are equal to or greater than 0.055 between Gli23PDD homozygotes and wild type or Gli23PDD heterozygotes within each age group.

To characterize Gli23PDD mutant mice further, we examined neural tube patterning of the mutant embryos and compared it with that of wt and Gli2lzki null mutants (Bai and Joyner, 2001). Shh is expressed in notochord and floor plate in the developing neural tube, and it specifies five ventral neural cell types in a concentration-dependent manner: V3, motoneurons (MNs), V2, V1, and V0 (Briscoe et al., 2000). Indirect immunofluorescence showed that the floor plate in Gli23PDD mutant, which is marked by Foxa2 expression, was similar to that of wt neural tube (Fig. 3S–T). The specification and patterning of Nkx2.2 positive V3 progenitors, Isl1 expressing motoneurons, Lhx3 expressing V2, En1 positive V1, and Evx1 expressing V0 neurons in Gli23PDD mutant were also indistinguishable from those in wt neural tube (Fig. 3, compare A to B, D to E, G to H, J to K, and V to W). Similarly, dorsal Pax6- and Pax7-expressing domains in Gli23PDD mutant, which are normally inhibited by Shh signaling, appeared to be similar to those in wt neural tube (Fig. 3, compare M to N and P to Q). In contrast, as reported previously (Ding et al., 1998; Matise et al., 1998), the floor plate and most of the Nkx2.2 positive domain in Gli2lzki null mutant were missing so that both lateral Lhx3 positive domains and Isl1-expressing motoneurons appeared to be fused together (Fig. 3C, F, I, U). The loss of Gli2 function also led to a ventral expansion of the Pax6 positive domain, although the ventral expansion of Pax7+ cells was not obvious (Fig. 3, compare M to O and P to R). Taken together, these data indicate that changes in the ratio of Gli2FL to Gli2Rep in Gli23PDD mutant does not lead to a detectable defect in neural tube patterning.

Figure 3. The neural tube patterning of Gli23PDD mutant is normal.

Figure 3

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Cross-sections of E10.5 mouse embryos at the forelimb position were stained with antibodies to the proteins shown above the panels or to the left for En1. The genotypes are indicated to the left or above the panels for En1. No difference was noticed for all the markers examined.

A Change in the balance between the Gli2FL activator and its repressor reduces the Hh pathway activity

To directly determine whether the change in the ratio of the Gli2FL to Gli2Rep in Gli23PDD mutant alters the Gli2 activity, we examined the expression of Gli1 and Ptch1 genes, the direct Hh targets, by whole mount in situ hybridization. As reported previously, both Gli1 and Ptch1 were expressed in spinal cord and posterior region of the limb bud around the Shh-expressing region in wt E10.5 mouse embryos (Hui et al., 1994; Goodrich et al., 1996). The levels of Gli1 and Ptch1 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 Gli2lzki null mutant (Fig. 4, compare C to A–B, F to D–E, K to G and I, L to H and J, and O to M and N). Thus, the change in the ratio of Gli2FL to Gli2Rep in Gli23PDD mutant indeed reduces Gli2 transcriptional activity.

Figure 4. The Hh pathway activity is reduced in Gli23PDD mutant.

Figure 4

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Whole mount (A–L) and spinal cord tissue section (M–O) in situ hybridization of E10.5 mouse embryos showing that the expression of both Gli1 and Ptch1 was reduced in Gli23PDD and Gli2lzki mutants as compared to that of wt. (G–L) Forelimb buds with anterior to the top.

To quantitatively compare the transcriptional activity of Gli23PDD with that of wt Gli2, we performed a cell based reporter assay using a Gli-dependent luciferase reporter (Sasaki et al., 1997) in Gli2lzki/lzki; Gli3Xt/Xt null mutant MEFs. The double mutant MEFs were chosen because the presence of endogenous Gli2 and Gli3 may mask the difference in the activity between Gli2 and Gli3PDD. Overexpression of Gli2 activates the reporter about 10 fold, while Gli23PDD overexpression increased the reporter activity only about 6 fold (Student’s t-test, n = 3, P<0.0118). The Gli23PDD transcription activity is higher than wt Gli3 (6 vs. 4 fold on average), although it is not statistically significant (n = 3, P < 0.2752)(Fig. 5). These results are in agreement with the reduced expression of endogenous Gli1 and Ptch1 expression revealed by whole mount in situ hybridization (Fig. 4).

Figure 5. Gli23PDD exhibits the lower transcriptional activity than wt Gli2.

Figure 5

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Gli2lzki/lzki; Gli3Xt/Xt mutant MEFs were transfected with an empty vector or indicated expression constructs together with a Gli-dependent firefly luciferase reporter and a TK-renilla luciferase (a transfection control). Two days later, luciferase activities were measured, and firefly luciferase activity was normalized against the renilla luciferase activity. The graph represents the normalized firefly luciferase activity from three independent experiments (n = 3, Student’s t-test P = 0.0118 between Gli2 and Gli23PDD, 0.2752 between Gli23PDD and Gli3, and 0.0034 between control and Gli3).

Gli23PDD processing is regulated by Hh signaling

If Gli23PDD full-length activity and its processing are still regulated by Hh signaling, the Gli2Rep levels could be significantly reduced in cells receiving Hh signals so that the mutant phenotypes would be alleviated. This may in part explain why the Gli23PDD mutant exhibits the minor phenotypes. We thus investigated whether Gli23PDD processing responded to Hh signaling. Wild type and Gli23PDD/3PDD MEFs were incubated with a Smo agonist, SAG, or a control vehicle. The processing of both Gli2 and Gli3 in the MEFs was determined by immunoblotting with Gli2 and Gli3 antibodies, respectively (Wang et al., 2000; Pan et al., 2006). As shown previously, Gli3 processing was efficiently (although incompletely) inhibited by treatment of MEFs with SAG (Fig. 6B), indicating that the MEFs responded to SAG stimulation. Similarly, Gli23PDD processing was also inhibited as indicated by the decrease in the Gli2Rep levels upon SAG treatment, although the inhibition was not as efficient as that of Gli3 processing. The treatment with SAG also significantly increased the levels of the Gli2FL activator (Fig. 6A). It should be noted that Gli2Rep in wt MEFs was not detected because it was likely to co-migrate with a background band that is slightly greater than Gli2Rep from Gli23PDD mutant (Fig. 2D). Together, these data indicate that Gli23PDD processing is inhibited by Hh signaling.

Figure 6. Gli23PDD processing is inhibited by Hh signaling.

Figure 6

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Immunoblots showing that the processing of endogenous Gli23PDD (A) and Gli3 (B) was inhibited by the treatment with SAG. WT, Gli23PDD/3PDD, and Gli2lzki/lzki; Gli3Xt/Xt double mutant MEFs were incubated with SAG, or DMSO vehicle overnight. The cells were lysed and subjected to immunoblotting with anti-Gli2, Gli3, or αtubulin antibodies. Arrows indicate Gli2FL, Gli2Rep, Gli3FL, and Gli3Rep. The ratio of Gli2FL to Gli2Rep and Gli3FL to Gli3Rep is quantified in C.

Discussion

Although both Gli2 and Gli3 are proteolytically processed to repressors in vivo, the extent of their processing is very different (Pan et al., 2006). We have previously shown that in cultured cells the PDD domain determines the differential processing and the site of Gli2 and Gli3 processing (Pan and Wang, 2007). However, it is not clear whether this is also true in vivo. It is also not known whether the level of Gli2 processing is important for the Gli2 function. In the present study, we demonstrate that Gli2 and Gli3 processing and the site of their processing are indeed controlled by PDD domain in vivo. We also show that changes in the ratio of the Gli2FL to the Gli2Rep in Gli23PDD mutant slightly reduce Gli2 net activity. These results indicate that the proper level of Gli2 processing is important for its activity and thus Hh signaling.

The PDD is functionally believed to serve as a ‘stop signal’ to prevent the proteasome from completely degrading Gli2 and Gli3. This leads to the site-specific and limited degradation of Gli2FL and Gli3FL to generate Gli2 and Gli3 repressors (Pan and Wang, 2007). A similar mechanism has also been reported for the Drosophila homolog of Gli proteins, Ci, but these studies are all based on overexpression (Tian et al., 2005; Wang and Price, 2008). In this study, we genetically engineered the Gli23PDD allele that expresses a Gli23PDD mutant protein in which Gli2 PDD is replaced by the Gli3 corresponding sequence. This chimeric protein is processed not only much more efficiently than wt Gli2 but also at a different position (Fig. 1D). These results indicate that PDD determines the levels and positions of Gli2 and Gli3 processing in vivo.

Mice homozygous for Gli23PDD are overall normal and fertile. However, they are on average slightly smaller than their wt siblings. The difference is consistent, albeit not statistically significant. Therefore, we believe that this is a phenotype resulted from the Gli23PDD mutation. Although the reason for this is not known, this phenotype is likely due to the reduced Gli23PDD transcriptional activity, as a previous study showed that a reduced Hh signaling results in smaller body size in mice (Milenkovic et al., 1999). Analysis of neural tube patterning does not reveal any difference between wt and the mutant. In contrast, the decrease in the Gli1 and Ptch1 expression in Gli23PDD mutant spinal cord and limb bud is evident (Fig. 4). The discrepancy between the neural tube patterning and the expression of Gli1 and Ptch1 in Gli23PDD mutant suggests that the former is less sensitive to an increase in the Gli2 repressor levels than the latter is. Nevertheless, the reduced Gli1 and Ptch1 expression in Gli23PDD mutant indicates that the net Gli2 activity is reduced. This reduction is likely the consequence of the change in the ratio of Gli2Rep to the full-length Gli23PDD, but not in the full-length Gli23PDD transcriptional activity 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.

Gli2 is generally considered to be a transcriptional activator in Hh signaling. This is consistent with the low level of Gli2 processing in vivo (Pan et al., 2006). However, whether the low level of the Gli2 repressor plays any role in Hh signaling is debatable. The observation that expression of Gli1 activator from the Gli2 locus can largely rescue the Gli2 mutant phenotypes supports the notion that Gli2 functions solely as an activator (Bai and Joyner, 2001). However, several lines of evidence suggest that Gli2 exhibits a weak repressor activity. First, we show that a small fraction of Gli2FL is processed to generate a Gli2 repressor (Pan et al., 2006). Second, Gli2P1–4 mutant embryos, which lack the Gli2 repressor due to mutations in four PKA sites in the Gli2 C-terminus, exhibit severe gain-of-function phenotypes (Pan et al., 2009). Third, Shh targets in Gli2+/−; Gli3Xt/Xt presomitic mesoderm are more repressed than those in Gli2−/−; Gli3Xt/Xt mutant (Buttitta et al., 2003; McDermott et al., 2005). Last, the present study shows that an increase in Gli2 repressor levels in Gli23PDD mutant results in a decreased Gli1 and Ptch1 expression (Figs. 4). The discrepancy of the two different conclusions is likely because the Gli2 repressor activity is not readily revealed as it overlaps with and is often masked by that of the Gli3 repressor.

Experimental procedures

Mouse strains and the generation of the Gli23PDD mutant knock-in allele

A PAC clone containing mouse Gli2 genomic DNA sequences (Geneservices, Inc. UK) was used to create a Gli23PDD targeting construct in the pGKneoloxP2DTA.2 vector (Soriano, 1997). The Gli2 construct was engineered by replacing the last third and second coding exons (encoding amino acid residues 585–751) of the mouse Gli2 gene with corresponding Gli3 cDNA sequence (648–814 aa) using recombinant DNA methods (Fig. 1A). The Gli23PDD construct was then introduced into W4 ES cells by electroporation, and neomycin (neo)-resistant clones were selected by incubating cells in ES cell growth medium containing G418 (200µg/ml). Targeted ES cell clones were identified by restriction enzyme digestion, followed by a Southern blot analysis of ES cell DNA using a 5'- and a 3'-probes. The 5'-probe identified a 5.84kb fragment in the targeted allele and a 5.36kb fragment in the wild-type (wt) allele following HindIII digestion. The 3' probe identified a 5.8kb fragment in the targeted allele and a 10.8kb fragment in the wt allele following EcoRI digestion (Fig. 1B). Two of the Gli23PDD targeted ES cell clones were injected into C57BL/6 blastocysts to generate chimeric founders, which were then bred with C57BL/6 to establish F1 heterozygotes. Neo cassette was removed by crossing the mutant animals with Act-cre mice. PCR analysis was used for routine genotyping with the following primers: YP105fwd, 5’-GAG TGA GGT GTG ACA GAC CCG-3’ and YP106Rev, 5’-GTA AAC CGG CAT GTG CTC ATG-3’, which produced a 214bp fragment for wt and 248bp for the mutant allele. Some times, the mutant allele was genotyped by using YP105fwd and CW649-loxpRev primer: 5’-CGA AGT TAT ATT AAG GGT TCC G-3’, which produced a 240bp fragment.

Gli2lzki mutant mice were obtained from Alexandra Joyner (Bai and Joyner, 2001). The mutant allele was genotyped with primers: BW575F, 5’-ATT GGT GGC GAC GAC TCC TGG A-3’ and BW575R, 5’-CAA TGA GAA CAG ACT AGA CCC T-3’, which produced a 200bp fragment. The wt allele was genotyped with primers: forward primer, 5’-ATA AAC CCA GCG TGC CTC CCA GAT GAC AGG-3’, and reverse primer, 5’-ATG GAG ACT TCT GCC CCA GCC CCT GCA CTG-3’, which produced a 300bp fragment. Gli3Xt/Xt mice were genotyped as described (Wang et al., 2007). All mice used in this study were in an either Swiss Webster (SW), 129v, and C57BL/6 or 129v and C57BL/6 mixed background.

Cell culture, generation of mouse embryonic fibroblasts (MEFs), and reporter assay

MEFs were grown in DMEM containing 10% fetal bovine serum (FBS), penicillin, and streptomycin (Invitrogen). WT, Gli23PDD/3PDD, and Gli2lzki/lzki; Gli3Xt/Xt MEFs were obtained from E13.5 mouse embryos. The MEFs were immortalized by passing them in DMEM containing 15% FBS until restoration of the normal growth rate. Thereafter, the cells were grown in the same medium with 10% FBS. Transfection of Gli2lzki/lzki; Gli3Xt/Xt MEFs were transfected using Fugene 6 reagent as described (Pan et al., 2006)(Roche). To determine Gli2 and Gli3 processing in response to SAG stimulation, MEFs were incubated with DMEM/10% FBS supplemented with SAG (300 nM) or DMSO vehicle (1/1000 (v/v)) for 24 hrs. Reporter assay was performed as described (Wang et al., 2000).

Immunoblotting, in situ hybridization, and immunohistochemistry

Detection of Gli2 proteins in E10.5 mouse embryos was performed by enrichment for Gli proteins with a Gli-binding oligonucleotide followed by immunoblotting with a Gli2 antibody, while protein lysates prepared from MEFs were directly used to detect endogenous Gli2 and Gli3 proteins as described (Pan et al., 2006). In situ hybridization of cryo-tissue sections and whole mount embryos were performed as described (Wang et al., 2007; Pan et al., 2009).

Indirect immunofluorescence of E10.5 mouse embryonic sections was preformed as described (Pan et al., 2009). Antibodies used for this study include: Foxa2, Nkx2.2, Isl1, Lhx3 (Lim3), En1, Evx1, Pax7, monoclonal antibodies (Developmental Study Hybridoma Bank (DSHB), Iowa); Pax6 (Covance). The secondary antibodies were obtained from Jackson ImmunoResearch laboratories.

Acknowledgements

We thank Dr. Alexandra Joyner for Gli2lzki mice. Isl1, Lhx3, Pax7, Nkx2.2, En1, Evx1, and Foxa2 monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa, Department of Biological Sciences, Iowa City, Iowa 52242, under contract NO1-HD-7-3263 from the NICHD. This study was supported by an NIH grant (R01GM70820) to B.W.

Support: NIH grant R01GM70820.

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