, 2001, Mizuguchi et al., 2001 and Lee et al., 2005). We confirmed this ability of Olig2WT to induce ectopic MNs (

Figures 4H–4J) but found that Olig2S147A was inactive in this regard ( Figures 4K–4M). Taken together, the data strongly suggest that S147 phosphorylation is necessary for the MN-inducing Decitabine function of OLIG2. In the spinal cords of Olig2 null mice, expression of the OLP markers PDGFRa and SOX10 is completely absent ( Lu et al., 2002, Takebayashi et al., 2002 and Zhou and Anderson, 2002), demonstrating an essential role for OLIG2 in OL lineage specification. In our Olig2S147A mutant mice, SOX10- and PDGFRa-expressing OLPs were missing at E14.5 ( Figures 5A–5D) but appeared later at E18.5, though in reduced numbers (∼15%) relative to Olig2WT controls ( Figures 5E, 5F, 5H, and 5I). At the time of their HDAC inhibitor first appearance, OLPs were scattered through all regions of the cord, not concentrated in the ventral cord as in wild-type mice. This is consistent with the demonstrated loss of the ventral pMN domain ( Figure 4)—the

source of most but not all OLPs in the cord—and suggests that S147 phosphorylation is not required for OLP specification from other progenitor domains that do not rely on the prior neuroepithelial patterning function of OLIG2. We further investigated the effect of S147

phosphorylation on OLP differentiation into myelin-forming OLs by culturing primary E18.5 spinal cord cells under conditions permissive for OL differentiation. (It was not possible to study OL differentiation next in vivo since Olig2S147A mutants die at birth due to the lack of MNs.) Myelin basic protein (MBP)-positive OLs formed in these mutant cell cultures as in wild-type cultures, demonstrating that S147 phosphorylation is not absolutely required for OL lineage progression ( Figure S4). To examine OLP-inducing activity further, we electroporated Olig2WT or Olig2S147A expression vectors into chick neural tube. It has been reported that Olig2WT can induce expression of OLP markers in the dorsal neural tube, after a delay of around 4 days post-electroporation ( Liu et al., 2007). We found that forced expression of Olig2S147A induced dorsal SOX10 expression well ahead of this schedule at 48 hr post-electroporation ( Figures 5M and 5N). As expected, Olig2WT did not induce SOX10 on this time scale ( Figures 5K and 5L). Taken together, our data suggest that OL fate is favored, even accelerated, when OLIG2 is not phosphorylated on S147. To investigate further the role of OLIG2-S147 phosphorylation in neural fate determination, we turned to an in vitro assay using P19 cells.