Knockdown of Sox6. Chicken embryos at HH stage 14–16 were used for transfection by electroporation of pSuperior shRNA-expressing plasmids. Embryos were harvested 48 h later, and electroporation was confirmed by immunostaining for GFP (A, E, and I). cSox6-sh1 and cSox6-sh2 express shRNAs specifically targeting cSox6 mRNA whereas the control plasmid Luc-shRNA is designed to target luciferase mRNA. RNA in situ hybridization was used to examine the expression of Sox6 (B, F, and J) and Sox2 (C, G, and K). Down-regulation of Sox6 is seen only in the cases of specific targeting (B and F) but not in the control case (J). Down-regulation of Sox2 was seen only in the cases of specific targeting of Sox6 (C and G) but not in the control case (K). Mis-expression of Isl1/2 in the ventricular zone was seen only in the cases of specific targeting of Sox6 (D and H) but not in the control case (L). Serial sections at the forelimb level were used.