Figure 2. Xist induction from the X^ΔTsix maternal X-chromosome in E6.5 extra-embryonic cells.

(a) RNA FISH detection of Xist (white), Tsix (green), and Pgk1 (red) RNAs in E6.5 extra-embryonic cells. Nuclei are stained blue with DAPI. Dashed boxes mark representative nuclei. Scale bar, 10 µm. (b) Quantification of Xist, Tsix, and Pgk1 RNA expression patterns. The X-axis of each graph represents the % nuclei in each class out of 100 total nuclei counted per genotype (from n > 3 embryos per genotype). Diagrams along the Y-axis depict all observed expression patterns. +, RNA expression detected from a single X-chromosome; + +, RNA expression detected from both X-chromosomes; -, absence of RNA detection. Pairwise comparisons of the frequency of individual gene expression patterns between wild-type and X^ΔTsix mutant embryos were performed using Fisher’s exact test. *, 0.001<p<0.01; **, p ≤ 0.001. Extra-embryonic cells show significantly increased level of inactivation of the X^ΔTsix X-chromosome (p=0.0003 for males; p=3.5×10⁻⁵ for females). (c) RT-PCR detection of Xist and Tsix RNAs in extra-embryonic tissues from individual E6.5 embryos. Results from three individual embryos of each genotype are shown. M, marker; NTC, no template control; +, RT; -, no RT control lane. (d) Sanger sequencing chromatograms of Xist RT-PCR products. Highlights mark a single nucleotide polymorphism that differs between the maternal X^Lab / X^ΔTsix alleles and the paternal X^JF1 allele. X^LabX^JF1 females express Xist only from the paternally-inherited X-chromosome, while X^ΔTsixX^JF1 females express Xist biallelically in extra-embryonic tissues. X^ΔTsixY embryos variably express Xist from the maternally-inherited X-chromosome.