Figure 4.

Contribution of Sox11 to Gdf5 expression in developing limbs. (A) mRNA levels of Col2a1, Sox6 and TenasinC (Tnc) in ATDC5 cells that were retrovirally transfected with a control vector (Rx-EV) or human SOX11 (Rx-SOX11) and the short hairpin RNA directed against GFP (Rx-shGFP) or against mouse Sox11 (Rx-shSox11). mRNA levels were determined by quantitative RT-PCR and expressed as means (bars) ± SDs (error bars) for 4 wells/construct. *P < 0.05 vs. Rx-EV. ^#P < 0.05 vs. Rx-shGFP. (B) Alcian blue staining of micromass cell cultures and mRNA levels of Gdf5 and SOX11 in those cultures after infection of RCAS virus encoding human SOX11. *P < 0.05 vs. RCAS-EV. (C) Whole mount in situ hybridization for Sox11 and Gdf5 in wild type chick embryos at HH stage 32 or 35 and in embryos after injection of RCAS virus encoding GFP-fused chick Sox11 at HH stage 10. The GFP fluorescence after the injection is also shown. Red arrows indicate the injected right hind limbs, while white arrows indicate the uninjected left hind limbs for control. Scale bars, 1 mm. (D) mRNA levels of chick Sox11, and Gdf5 genes of hind limbs at HH stage 35 (E8.5) after the RCAS injection of GFP-fused chick Sox11. The uninjected left hind limb is used as control. *P < 0.05 vs. control. (E) Western blotting for Gdf5 and β-Actin (ACTB) in HH stage 35 (E8.5) chick hind limbs after the RCAS injection of GFP-fused chick Sox11. The uninjected left hind limb is used as control. The GFP fluorescence in the embryos used for Western blotting analysis is also shown. Red arrow indicates the injected hind limb and white arrow shows the control hind limb. Densitometric analysis was performed with Image J software.