Collectively, these data establish a functional hierarchy between Sox9 and NFIA during the initiation of gliogenesis, where the ability of Sox9 to promote the initiation of gliogenesis is linked to its direct induction of NFIA expression. The foregoing data gathered in the embryonic chick spinal cord indicate that Sox9 directly regulates NFIA induction and that this relationship is crucial for the initiation of gliogenesis. Histone Acetyltransferase inhibitor We next sought

to determine whether these same regulatory relationships are present in the mouse. First, we determined the temporal patterns of Sox9 and NFIA induction and found that Sox9 is induced prior to NFIA in the VZ of the embryonic spinal cord (Figures 2R–2Y). Examination of the mouse e123 enhancer revealed a Sox9 site within the conserved Sox9-Mu2 region (Figures 1B and 1C), and therefore we next determined whether Sox9 could ChIP this site NLG919 in the e123 enhancer region within the endogenous mouse NFIA promoter. To this end, we performed ChIP from E12.5 mouse spinal cord and found that Sox9 is capable of interacting with the Sox9-Mu2 binding site in the e123 enhancer of the mouse NFIA promoter (Figure 1CC). These data suggest that Sox9 and NFIA have a similar regulatory relationship in mouse and chick. To provide genetic evidence

linking Sox9 to the induction of NFIA during the initiation of gliogenesis, we intercrossed the Sox9fl/fl and nestin-cre

mouse lines ( Akiyama et al., 2002). This approach has been used previously to conditionally delete Sox9 in VZ populations of the embryonic spinal cord and revealed a delay in the generation of oligodendrocytes ( Stolt et al., 2003). Given the regulatory relationship between Sox9 and NFIA, we reasoned that loss Thalidomide of Sox9 in this context would impact the timing and/or the expression of NFIA. To examine this possibility, we generated E11.5–E12.5 Sox9fl/fl;nestin-cre and Sox9fl/+;nestin-cre embryos and assessed the expression of NFIA ( Figures 2Z–2GG). NFIA is normally induced in the VZ of the spinal cord at E11.5, but in the absence of Sox9, induction of NFIA was delayed by 1 day to E12.5 ( Figures 2DD–2GG, arrow). Analysis at E12.5 revealed reduced levels of NFIA expression in the absence of Sox9 ( Figures 2FF and 2GG, arrow). Further analysis of these embryos revealed that the induction of GLAST is also delayed from E11.5 to E12.5 and reduced in the absence of Sox9 ( Figures 2HH–2KK), correlating the expression patterns of NFIA and GLAST and reinforcing the functional hierarchy established in our chick studies. These mouse studies provide genetic evidence that Sox9 is necessary for the induction and expression of NFIA during the initiation of gliogenesis in the developing spinal cord.