Deletion of Sox9-Mu2 resulted in a loss of e123 activity at E6, indicating that this site mediates e123 activity (ΔMu2-GFP) (Figures 1X and 1BB). Further supporting the regulatory relationship between e123 and Sox9, coelectroporation of e123 with a dominant-negative version of Sox9 (Sox9-EnR) resulted in a loss of activity at E6 (Figure S2; Scott et al., 2010). Next, we performed chromatin Panobinostat manufacturer immunoprecipitation (ChIP) assays to determine whether Sox9 directly associates with the Mu2 site in e123 region of the endogenous NFIA promoter. To this end we electroporated HA-Sox9 into the embryonic chick

spinal cord, harvested embryos at E4, and performed ChIP assays on chick spinal cord lysates. As indicated in Figure 1CC, Sox9 is able to specifically ChIP the Sox9-Mu2 site in the e123 enhancer of the NFIA promoter. Taken together, these data indicate that Sox9 is necessary and sufficient for the activity of the e123 enhancer and does so via a direct mechanism. Because Sox9 directly controls e123 enhancer activity, we reasoned that manipulation of

Sox9 activity would impact expression of NFIA. To this end we introduced a dominant repressor form of Sox9, Sox9-EnR NVP-BKM120 order (Scott et al., 2010), into the chick spinal cord and found that it inhibited the expression of NFIA (Figure 2F). Next we introduced wild-type Sox9 or a dominant activator form of Sox9, Sox9-VP16, and found that both forms are sufficient to induce ectopic NFIA expression in regions outside the VZ (Figures 2G, 2H, and 2P, arrows). These observations indicate that Sox9 functions as a transcriptional activator to induce NFIA expression and are consistent with our findings that it regulates the activity of the e123 enhancer. In the course of analyzing the Sox9 and the Sox9-VP16 electroporated embryos, we noticed that in regions outside the VZ demonstrating ectopic

NFIA expression, there was also ectopic old expression of the early astro-glial precursor marker GLAST (Figures 2L, 2M, and 2Q, arrows; Shibata et al., 1997). This observation indicates that Sox9 and Sox9-VP16 are sufficient to induce ectopic expression of glial precursor markers and is consistent with a role for Sox9 during the initiation of gliogenesis. Given that these GLAST-expressing regions contain ectopic NFIA and that NFIA is necessary for GLAST expression, we next determined whether the ability of Sox9 to induce ectopic GLAST is reliant upon its regulation of NFIA (Deneen et al., 2006). Here, we coelectroporated Sox9-VP16 along with an NFIA-shRNAi and examined the expression of GLAST and a set of other astro-glial precursor markers (Figure S3). As shown in Figures 2I, 2N, and 2Q, Sox9-VP16 is not capable of inducing ectopic GLAST in the absence of NFIA, indicating that Sox9 regulation of NFIA results in the ectopic induction of glial precursor markers.