SHH activates GLI3 (and GLI2) by inducing its proteolytic conversion from a full-length transcriptional activator into a truncated N-terminal repressor ( Ruppert et al., 1990, Dai et al., 1999 and Wang et al., 2000). It is believed that PKA phosphorylation stimulates GLI3 cleavage ( Wang et al., 2000 and Tempe et al., 2006), which might underlie

the repressive action of PKA on SHH signaling since truncated GLI3 represses the SHH pathway ( Dai et al., 1999, Wang et al., 2000, Bai et al., 2004 and Tempe et al., 2006). GLI3 is not the only component of the SHH pathway that can be PKA phosphorylated, and PKA has been shown to Protein Tyrosine Kinase inhibitor play a positive as well as a negative role in SHH signaling. For example, in Drosophila, PKA phosphorylation of SMO, the Hedgehog (HH) coreceptor, promotes SMO accumulation on the primary cilium and triggers HH pathway Ku 0059436 activation ( Jia et al., 2004). Also, during limb development, elevating PKA activity by Forskolin treatment or by infecting with a retroviral PKA expression vector exerts a positive effect on SHH signaling, resulting in an altered pattern of digits ( Tiecke et al., 2007). We examined the effect of stimulating the PKA pathway in neonatal mouse cortical cell cultures

with Forskolin and dibutyryl cyclic AMP (db-cAMP), a cell-permeable analog of cAMP, and found that the number of NG2-positive OLPs was significantly decreased compared to untreated cultures (Figure S7). This is consistent with expectation since our other data predict that elevating PKA activity should increase OLIG2-S147 phosphorylation and stimulate neurogenesis at the expense of OLPs. However, in the light of the above discussion, it is clear that PKA probably has multiple parallel functions, and our experiments with Forskolin/db-cAMP should be interpreted cautiously. Our data raise the obvious question: What is the key event that signals S147 dephosphorylation and triggers the MN-OLP switch in pMN? Notch signaling is known to play an important role in glial cell development in the CNS (Wang et al., 1998, Park and Appel, 2003, Park et al., 2005 and Deneen et al., 2006). over Constitutive activation

of components of the Notch pathway in chick spinal cord can downregulate NGN2 expression in pMN and initiate OLP generation (Zhou et al., 2001). Notch1 is expressed by neuroepithelial cells throughout the neural tube, and its ligand Jagged-2 is expressed exclusively in the pMN domain of zebrafish spinal cord during late neurogenesis (Yeo and Chitnis, 2007). These and other observations frame the Notch pathway as a potential key player in the MN-OLP switch. It is possible that activated Notch-1, via its effector HES5, might induce expression of specific phosphatases and/or repress phosphatase inhibitors, resulting in dephosphorylation of OLIG2-S147 and initiation of OLP production. It will be worth exploring these ideas in the future.