Figure 6

The identification of direct and indirect candidate targets of EBF3 by RT-QPCR. hGR-XEBF3 mRNA and Noggin mRNA were injected into one-cell stage embryos, and animal caps were collected at the blastula stage (stage 9). The animal caps were divided into four groups, based on CHX and DEX treatment: -C-D, -C+D, +C-D, and +C+D. After a 3.5-hour incubation with CHX and/or a 3-hour incubation with DEX, total RNA was isolated from each animal cap group. RT-QPCR was conducted with the isolated total RNA. The expression level was normalized with the expression level of histone h4 and then normalized to the expression level of -C+D, for each gene, at 100 arbitrary units. (A-C) The expression levels of all candidate target genes in controls (-C-D and +C-D) are very low compared to the DEX-treated condition of -C+D. (A) The expression level of peripherin in +C+D (61%) is only partially reduced compared to -C+D, indicating that the majority of its expression is controlled by EBF3 directly. (B) The expression level of pcdh8 in +C+D (5%) is much lower than in -C+D and is similar to the levels of the control conditions, indicating that it is an indirect candidate target. (C) The expression level of aml1 in +C+D (37%) is lower than the expression level in -C+D but higher than levels of the control conditions, indicating that its expression is partially controlled by EBF3 directly. Error bars represent standard error of the mean. The results for the remaining candidate target genes are shown in Additional file 6. N = 3 replicates, 20 to 30 animal caps per condition.