Fig. 4: Oct4 is downregulated prior to NCC formation.

A Oct4-EGFP expression (green) at E7.5 (n = 5) and E8.5 (n = 5). Oct4 is highly expressed in the embryo proper at E7.5 according to high GFP expression but is downregulated by E8.5 as evidenced by the absence of GFP expression. B Oct4-EGFP and SOX10 expression in Nr6a1 null and control littermates. Oct4 is exogenously expressed in the null compared to control embryos which do not display Oct4 (GFP) expression. Notably, the pattern of SOX10 in the null suggests Oct4 is not expressed in the NCC that have managed to form. C Quantification and statistical analysis of Oct4-EGFP expression within NCC and neuroepithelial cells (NE) in Nr6a1 null (n = 3) and control littermates (n = 4) depicted by a boxplot. One-way ANOVA determined a statistical significance of <0.0001 across the cell populations. No statistical significance was found by unpaired parametric (two-tailed) t-test when comparing the control NCC and Nr6a1 null populations (difference between means is −5.692 + /- SEM 14.75) or the control NCC and control NE populations (difference between means is 15.62 + /- SEM 11.96). The Nr6a1 null embryos had a statistically significant increase in Oct4-EGFP expression in the NE cells compared to the NCC of the null that were SOX10+ (p = 0.0033, difference between means is 151.1 + /- SEM 24.14)) and compared to control NE cells (p = 0.0012, difference between means is 129.8 + /- SEM 19.77). This data demonstrates the maintenance of Oct4 expression in the Nr6a1 null and suggests that the SOX10+ cells in the null do not have Oct4 expression, consistent with the requirement for Oct4 downregulation in NCC formation. The bounds of each sample set are as follows: Control NCC (min=142.777, max=179.966, mean=165.8), Nr6a1 −/− NCC (min=138.352, max=184.154, mean=160.1), Control NE (min=168.913, max=206.145, mean=181.4), and Nr6a1 −/− NE (min=272.514, max=340.373, mean=311.2).