The first clear defect was a decrease in N-cadherin staining starting around 12 hr posttransfection,

followed thereafter by a loss of Sox2 staining and cytoplasmic accumulation of Numb at 24 hr posttransfection, and the ectopic formation of NeuN+ neurons within the VZ by 36 hr posttransfection (Figures 4A–4O). We did not observe any notable elevation of either Ngn2 or NeuroM above that already present in the spinal cord during this time course (data not shown), suggesting that the prodifferentiation actions of Foxp4 work downstream or in parallel with endogenous proneural gene activity. We next FACS-isolated transfected cells from the electroporated spinal cords and measured mRNA expression levels using quantitative selleck screening library PCR. Foxp4 misexpression resulted in an ∼45% decrease in N-cadherin mRNA within 6 hr and

an ∼65% decrease by 12 hr postelectroporation ( Figure 4P). We did not observe any significant decrease in the expression of other AJ genes such as β-catenin, Obeticholic Acid in vitro Cdc42, RhoA, and aPKCζ at the 6 hr time point, though β-catenin mRNA was moderately reduced by 12 hr postelectroporation ( Figure 4P). Despite this latent β-catenin reduction, we did not detect any changes in β-catenin activity as measured by a cotransfected Wnt/β-catenin-responsive reporter, TOP-dGFP ( Dorsky et al., 2002), or find any correlation between reporter expression and the endogenous pattern of Foxp4 expression ( Figures S2S–S2V). These results suggest that the decline in β-catenin levels may be secondary to N-cadherin loss. In evaluating the expression of other genes, we found that Foxp4 potently suppressed Sox2 mRNA by ∼70% within 6 hr postelectroporation Dipeptidyl peptidase ( Figure 4P). Despite this early transcriptional effect, Sox2 protein did not decline until ∼18–24 hr postelectroporation, at which time N-cadherin was undetectable ( Figures 4A, 4B, 4F, and 4G). Together, these data indicate that Foxp4 can rapidly suppress both N-cadherin and Sox2 mRNA expression, but N-cadherin protein is more labile such that it declines

before Sox2 and thus initiates the process of neuroepithelial detachment. To confirm that Foxp4 directly regulates N-cadherin, we aligned the genomic sequence of the chick, mouse, and human Cdh2 (N-cadherin) loci and identified several evolutionarily conserved regions within introns 2 and 3 that contained canonical Foxp binding sites ( Figures 4Q and S6A–S6G). Foxp4 binding to these elements was measured through chromatin immunoprecipitation assays using differentiating MN progenitors produced in vitro from mouse embryonic stem cells as a proxy for spinal cord tissue. Foxp4 binding was prominent at a highly conserved element within intron 3 [i3a] but not at other sites tested ( Figures 4Q and S6).