Figure 4.
Knockdown of Tcf3 and activation of the Wnt pathway in mES cells reveal a role for Tcf3 in repression of target genes and a role in regulating pluripotency. (A) Tcf3 knockdown results in up-regulation of target genes. The effect of Tcf3 knockdown on gene expression was measured by hybridization of labeled RNA prepared from Tcf3 knockdown cells against RNA prepared from cells infected with nonsilencing control lentivirus at 48 h post-infection. A heat map of biological replicate data sets of expression changes was generated where genes are ordered according to average expression change. Tcf3 target genes have a higher average expression change than the average for all genes upon knockdown of Tcf3. (B) Wnt CM results in up-regulation of Tcf3 target genes. The effect of Wnt activation on gene expression was measured by hybridization of labeled RNA prepared from mES cells grown in Wnt CM against RNA prepared from cells grown in mock CM. A heat map of biological replicate data sets of expression change upon addition of Wnt CM where genes are ordered according to average expression change of replicates. Tcf3 target genes have a higher average expression change than the average for all genes upon addition of Wnt CM. (C) Tcf3 knockdown results in increased expression of Oct4, Sox2, and Nanog. Real-time PCR demonstrates that Oct4, Sox, and Nanog have increased expression upon knockdown of Tcf3. Values are normalized to Gapdh transcript levels, and fold change is relative to cells transfected with a nonsilencing hairpin. (D) Tcf3 knockdown results in increased staining for Oct4 and Nanog. Immunofluorecence was performed on mES cells grown one passage off of feeders that were either infected with Tcf3 knockdown lentivirus or infected with nonsilencing control lentivirus. Cells were fixed with 4% paraformaldehyde 96 h post-infection. Cells were stained with Oct4, Nanog, and DAPI. Images for Oct4 and Nanog staining were taken at 40× magnification and an exposure time of 300 msec. Tcf3 KD 1 and KD 2 represent different knockdown hairpin constructs. Tcf3 KD 2 is the virus also used in A, C, E, and F. (E) Tcf3 knockdown results in a significant increase of Oct4 staining. Quantification of Oct4 staining was performed in cells infected with Tcf3 or Gfp knockdown virus. (F) Tcf3 knockdown cells proliferate over more passages in the absence of LIF. Relative cell numbers of ES cells transfected with Tcf3 or control virus through multiple passages off of feeders in the presence or absence of LIF. Identical cell numbers were initially plated, and cells were split 1:12 every 2–3 d. Cells were counted at each passage and values for cells grown in the absence of LIF were normalized to cells grown in the presence of LIF.