Figure 2

Ectopic Xist RNA clouds observed in Tsix -mutant male ES cells. A. Percentage of cells containing Xist RNA clouds. Note the high percentage of Xist-positive cells in X^Δ/Y cells when differentiated in the presence of RA. Days of differentiation are listed as day 2 (d2), day 4 (d4), and day 6 (d6). Day 6 X^Δ/X EB+RA, not determined (n.d.). X/Y, wild-type male ES line; X/X, wild-type female ES line; X^Δ/Y, Tsix^ΔCpG/Y male ES line; X^Δ/X, Tsix^ΔCpG/+ female ES line; n > 200 for each timepoint and cell line examined. Error bars represent the standard deviation from three independent biological replicates. P was calculated by paired, two-tailed Student's t-tests. Differences in the number of Xist⁺ cells for RA-free versus RA-induced X^Δ/Y cells were statistically significant on all differentiation days (each red asterisk denotes P < 0.05 for day 2, day 4, and day 6 comparisons). EB, EB+RA, TC, and TC+RA conditions are described in the Materials and Methods. B. Xist RNA FISH showing ectopic and diffuse Xist cluster formation (arrows) in differentiating Tsix^ΔCpG male cells. Arrows denote Xist RNA clusters. The RNA FISH probe is double-stranded. Pinpoint signals primarily detect mildly elevated Xist expression in mutant cells caused by the Tsix deficiency, though ~10% residual Tsix expression from an upstream promoter may also contribute [13]. Below is a side-by-side comparison at day 6 (in grayscale) of a normal compact Xist RNA cloud (X/X, left) next to the ectopic dispersed Xist cluster observed in X^Δ/Y cells (right). C. X- and Y-chromosome paint confirm that all ES cell lines carry the appropriate number of sex chromosomes. Red, X-chromosome; green, Y-chromosome.