, 2003). However, OLIG2 and NKX2.2 are not tightly colocalized in the pMN domain in the run up to MN-OLP fate switching in mice (Sun et al., 1998 and Richardson et al., 2006), so it is not clear that OLIG2 and NKX2.2 normally work together in OLP specification. In fact, OLPs develop normally

in NKX2.2 null mice, although their subsequent differentiation into OLs is prevented (Qi et al., 2001). Another potential cofactor for OLIG2 is MASH1/ASCL1; initially (∼E12.5) there are reduced numbers of OLPs in MASH1 null spinal cords, but MAPK inhibitor this soon resolves, suggesting that MASH1 is not critical for OLP specification (Sugimori et al., 2008). We found that S147A mutation did not have a noticeable effect on binding of OLIG2 to either NKX2.2 or MASH1 (Figure S2A), so we probably have to look elsewhere for critical determinants of OL lineage specification in the spinal cord. The transcription factor NF1A has been proposed to trigger gliogenesis throughout the neural tube (Deneen et al., 2006), but its physical and functional relationships with OLIG2 have yet to be investigated. Recently, OLIG2 was shown to interact with the bHLH protein E47 to directly activate the Sox10-U2 promoter in the OL lineage ( Küspert et al.,

2010). The results of our present study suggest that dephosphorylated Regorafenib clinical trial OLIG2S147 might specifically interact with E47 in this context. Epigenetic modifications play an important part in cell fate decisions, including OLP specification. For example, in the absence of histone deacetylases 1 and/or 2 (HDAC1/2), OLP formation in the ventral spinal cord is disrupted (Cunliffe and Casaccia-Bonnefil, 2006, Ye et al., 2009 and Li et al., 2009). It will be important in the future to investigate the potential interactions between OLIG2, HDACs, and other histone modifiers (e.g., methylases) and how these interactions

might be influenced by posttranslational modification. Our study focused on reversible phosphorylation of OLIG2-S147, an evolutionarily conserved site that is predicted to be a PKA target. We provided evidence that OLIG2-S147 can be phosphorylated by PKA in cultured Cos-7 cells and that dnPKA inhibits phosphorylation. Despite this, we cannot conclude that PKA is the only—or even the primary—protein serine kinase to target S147 in vivo. For example, the consensus target sequence for PKA found (R[R/K]X[S/T]) can overlap with that of PKC ([R/K]X[S/T]U[R/K]) (U, hydrophobic). Indeed, PKA and PKC are thought to phosphorylate common acceptor sites on the bHLH proteins HAND1 and HAND2 (Firulli et al., 2003). Nevertheless, the fact that Olig2S147A mice failed to develop MNs suggests that PKA might normally play a positive role in MN development. This seems counter to the popular idea that PKA negatively regulates the SHH pathway ( Li et al., 1995, Ruiz i Altaba, 1999, Epstein et al., 1996, Hammerschmidt et al., 1996 and Jacob and Briscoe, 2003).