The expression of Gli3, regulated by HOXD13, may play a role in idiopathic congenital talipes equinovarus

Abstract

Background: Idiopathic congenital talipes equinovarus (ICTEV) is a congenital limb deformity. Based on extended transmission disequilibrium testing, Gli-Kruppel family member 3 (Gli3) has been identified as a candidate gene for ICTEV. Here, we verify the role of Gli3 in ICTEV development.

Methods: Using the rat ICTEV model, we analyzed the differences in Gli3 expression levels between model rats and normal control rats. We used luciferase reporter gene assays and ChIP/EMSA assays to analyze the regulatory elements of Gli3.

Results: Gli3 showed higher expression levels in ICTEV model rats compared to controls (P < 0.05). We identified repressor and activator regions in the rat Gli3 promoter. The Gli3 promoter also contains two putative Hoxd13 binding sites. Using EMSA, the Hoxd13 binding site 2 was found to directly interact with Hoxd13 in vitro. ChIP assays of the Hoxd13-Gli3 promoter complex from a developing limb confirmed that endogenous Hoxd13 interacts with this region in vivo.

Conclusion: Our findings suggest that HoxD13 directly interacts with the promoter of Gli3. The increase of Gli3 expression in ICTEV model animal might result from the low expression of HoxD13.

Figure 1

Semi-quantitative RT-PCR analysis of Gli3 mRNA expression in flexor hallucis longus and lung tissue. Gli3 expression was not found in the flexor hallucis longus. Lanes 1 is an RT-PCR sample from an ICTEV patient flexor hallucis longus, lane 2 is a normal control flexor hallucis longus RT-PCR and lane 3 is a normal control lung RT-PCR. β-actin expression is shown as a control for all samples. Lane M is a size marker.

Figure 2

Comparison of ICTEV and normal embryos at GD21. (A) Foot of an ICTEV patient, showing cavus, adductus, varus and equines. (B) The hindlimbs of normal embryos at GD21 presented with normal development. (C) The hindlimbs of ICTEV embryos at GD21 presented with dysplasia of the foot, showing cavus and adductus as observed in ICTEV patients.

Figure 3

Real-time PCR analysis of Gli3 expression in the hindlimb of ICTEV model rat and normal control rat embryos. Compared to the normal control, Gli3 mRNA expression was significantly enhanced. With birth drawing near, Gli3 expression in both model and normal rats had the tendency to decrease. Column 1, 3, 5 and 7 show Gli3 relative expression in ICTEV model rat at GD15, GD17, GD19 and GD21, respectively. Column 2, 4, 6 and 8 show Gli3 relative expression in normal control rat embryos at GD15, GD17, GD19 and GD21, respectively.

Figure 4

Western blot results. Western blots were used to determine Gli3 protein abundance in nuclear extracts from the hindlimb tissue of ICTEV model rat and normal control rat embryos. Compared to the normal control, Gli3 expression was significantly enhanced in model rats. With birth drawing near, Gli3 protein expression in both model and normal rats had a tendency to decrease. Lane 1 is the protein of ICTEV model rat embryos at GD15, lane 2 is the protein of normal rat embryos at GD15, lane 3 is the protein of ICTEV model rat embryos at GD17 and lane 4 is the protein of normal rat embryos at GD17.

Figure 5

Immunohistochemistry results. Immunohistochemistry revealed that Gli3 expression in the hindlimb tissue of ICTEV model rat embryos was higher than that in normal control rat embryos at GD19 (400×). (A) Hindlimb tissue of a normal control rat embryo labeled with PBS. (B) Hindlimb tissue of an ICTEV model rat embryo labeled with Gli3 antibody. (C) Hindlimb tissue of a normal control rat embryo labeled with Gli3 antibody.

Figure 6

Relative activities of rat Gli3 promoter regions in a luciferase reporter construct. Activities were measured in L6 cells. Findings suggested positive regulatory elements existed in the regions from -1107 to -532, -388 to -128 and from -128 to -48 upstream of Gli3 and negative regulatory elements in the region from -532 to -388 upstream of Gli3.

Figure 7

Potential binding sites. The rat Gli3 proximal promoter region contains two potential HoxD13 binding sites (gray boxes). The transcription start site is indicated by an arrow. The Gli3 protein translation start site (ATG) is indicated.

Figure 8

ChIP results. The ChIP assay of putative HoxD13 binding sites 1 and 2 in the Gli3 promoter. Only HoxD13 binding site 2 binds HoxD13 in vivo. In lanes 1 and 5, chromatin from rat embryonic hindlimbs was immunoprecipitated with the HoxD13 antibody. In lanes 2 and 6, the Sox9 antibody was used as a negative control. Lanes 3 and 7 show the enzymatic shearing before immunoprecipitation. Genomic DNA was used as a positive control (lanes 4 and 8). Lane M is a size marker.

Figure 9

EMSA results. Electrophoretic mobility shift assay (EMSA) using oligonucleotide probes containing the HoxD13 binding site 2. The *** denotes unbound biotin-labeled DNA, ** denotes Hoxd13-DNA complexes and * denotes Hoxd13-antibody-Hoxd13-DNA complexes.