Figure 3.

Males Undergo Transient XCI

(A) RNA FISH for Xist (red) in male 2iL ESCs at 1.5 days of differentiation in FA using a strand-specific probe. White arrowheads indicate Xist signals. Quantification of the different Xist RNA patterns is shown.

(B) Immuno-RNA FISH for Xist (red) and H3K27me3 or H3K27ac (green) in male 2iL ESCs at 1.5 days of differentiation in FA. White arrowheads indicate Xist cloud.

(C) RNA FISH for Xist (red) and Rnf12, Nexmif, or Huwe1 (grayscale) in male 2iL ESCs at 1.5 days of differentiation in FA.

(D) Quantification of RNA FISH patterns for the X-linked genes Rnf12, Nexmif, or Huwe1 and Xist as shown in (C). Gray indicates the presence of Rnf12/Nexmif/Huwe1 signal, and pink indicates the absence of Rnf12/Nexmif/Huwe1 signal.

(E) RNA FISH for Xist (red) in female 2iL ESCs at 1.5 days of differentiation in FA using ss probe. Quantification of different Xist RNA patterns is shown.

(F) RNA FISH for Xist (red) and Rnf12 or Huwe1 (grayscale) in female 2iL ESCs at 1.5 days of differentiation in FA.

(G) Quantification of RNA FISH patterns for X-linked genes Rnf12 or Huwe1 and Xist as shown in (F). Dark gray indicates biallelic Rnf12/Huwe1 signal, light gray indicates monoallelic Rnf12/Huwe1 signal, and pink indicates the absence of Rnf12/Huwe1 signal.

(H) Immuno-RNA FISH for Xist (red), NANOG (green), and OCT4 (grayscale) in male 2iL ESCs at 1.5 days of differentiation in FA. White arrowheads indicate Xist clouds.

(I) Percentage of NANOG- and OCT4-expressing cells in the population (left) and in cells exhibiting Xist cloud (right) as shown in (H).

Fisher’s exact test was used for statistical analysis. ESC lines used were XY1 and XX1. Scale bar represents 5 μm.