FIGURE 3.

Ovol2 knockdown phenotype is rescued by expression of shRNA-resistant mutant Ovol2. A, silent mutations in mut-Ovo that prevented their knockdown by Ovol2 shRNA-1. B, qRT-PCR analysis of total Ovol2 mRNA levels from ESCs with control (Ctrl) or Ovol2 shRNA-1 or shRNA-1 ESCs expressing the mutant Ovol2. The vectors carry RFP that was used for cell sorting. C, the three groups of ESCs described in B were induced in the SFEB condition for 6 days. The expression levels of Sox1 at differentiation day 4 and that of Tuj1 at day 6 were analyzed by qRT-PCR and normalized to the expression of Gapdh. The values for each marker in control EBs were designated as 1, respectively. D, immunostaining and quantification of Tuj1 positive cells in day 6 EBs cultured under the conditions described in C. E, EBs from the three groups of ESCs were cultured in unbiased differentiation medium for 8 days. The expression levels of Sox1 and Pax6 were analyzed by qRT-PCR. F, qRT-PCR analysis of the mRNA levels of T (at day 6) and of Flk1 and Sox17 (at day 8) in the EBs cultured under the conditions described in E. G, immunostaining and quantification of T-positive cells in day 6 EBs described in E. Scale bar for all of the images, 100 μm. Error bars, S.E.