In order to study the phenotypic consequences of OLIG2-S147 phosphorylation in vivo, we generated Olig2S147A mutant mice. We modified OLIG2-coding sequence in an Olig2 PAC clone (not containing Olig1), introducing the S147A mutation while simultaneously adding a V5 epitope tag to the C terminus ( Figure 3A). Transgenic mice were generated by pronuclear injection. V5-tagged Olig2S147A
and Olig2WT mice were made in parallel and single-copy founders of both lines were selected for further study ( Figure 3B). Immunofluorescence microscopy confirmed that the Olig2S147A and Olig2WT PAC transgenes were faithfully selleck chemicals llc expressed in the embryonic spinal cords of both lines ( Figures 3C and 3D). We subsequently removed selleck kinase inhibitor the endogenous Olig2 alleles by crossing the PAC transgenes into an Olig2 null background ( Lu et al., 2002), thereby obtaining single-copy Olig2S147A and Olig2WT lines (i.e., Olig2S147A:Olig2−/− and Olig2WT:Olig2−/−). For some experiments we also bred the PAC transgenics with Olig1/Olig2 double-null mice ( Zhou and Anderson, 2002), which express green
fluorescent protein (GFP) under transcriptional control of Olig2 (see below). Progenitors in p3, the ventral-most progenitor domain of the embryonic spinal cord, express the transcription factor NKX2.2, whereas progenitors in the p2 domain express IRX3 and a high level of PAX6 (Briscoe et al., 2000). The pMN domain lies between p3 and p2 and is marked by expression almost of OLIG2 and a low level of PAX6 (Lu et al., 2000 and Zhou et al., 2000; Figure 4A). OLIG2 is essential for establishing and
maintaining the pMN domain through cross-regulatory interactions with transcription factors in neighboring domains. For example, OLIG2 represses expression of Irx3 and Pax6 ; in the absence of OLIG2 function, Irx3 and Pax6 are derepressed in pMN, which takes on the character of p2, generating V2 interneurons and astrocytes instead of MNs and OLPs ( Lu et al., 2002 and Zhou and Anderson, 2002) ( Figure 4B). This can be regarded as a “homeotic” transformation pMN → p2. We found that the pMN domain specifically was missing in our Olig2S147A mice ( Figures 4C and 4D). Moreover, in Olig2S147A:Olig2 GFP/−, Olig1+/− embryos, most pMN precursors (marked by GFP) were observed to adopt a p2 fate (high PAX6 expression) ( Figure S3). These findings demonstrate that mutation of the S147 phosphate acceptor site destroys the neuroepithelial “patterning” function of OLIG2. It is known that MNs fail to develop in the spinal cords of Olig2−/− embryos as a consequence of losing the pMN progenitor domain ( Lu et al., 2002, Takebayashi et al., 2002 and Zhou and Anderson, 2002; Figure 4E). Similarly, our Olig2S147A mutant mice failed to generate MNs, judging by the lack of expression of the MN-specific HD transcription factor HB9 ( Figure 4G). HB9-positive MNs developed normally in Olig2WT mice ( Figure 4F).