Figure 7. MSL1 and MSL2 depletion leads to enhanced and chaotic Xist accumulation in early differentiation.

(A) RNA-FISH for Huwe1 (red) and Xist (green) in: scrambled control, shMsl1-, shMsl2-, and shMof-treated female ESCs. Nuclei were counterstained with DAPI (blue). White arrows denote foci corresponding to Huwe1 or Xist; dashed lines indicate nuclei borders. For additional images, phenotypes and quantifications see Figure 7—figure supplement 1B–D. For probe references see ‘Materials and methods’. (B) Expression analysis for Xist in undifferentiated, day 2 (D2) and day 3 (D3) differentiating female ESCs treated with scrambled RNA (shScram) or shRNA against Mof, Msl1, and Msl2. All results are represented as arbitrary units (Xist expression in undifferentiated ESCs = 1) normalized to expression levels in shScram (normalized to Hprt) and expressed as means ± SD in three biological replicates. p-values for D2-to-D3 expression change were obtained using unpaired t test. (C) RNA-FISH for Huwe1 (red) and Xist (green) in: scrambled control, shMsl1-, shMsl2-, and shMof-treated differentiating female ESCs. Nuclei were counterstained with DAPI (blue). RNA-FISH was performed on the sixth day of knockdown (after 72 hr of differentiation). Percentages indicate number of cells with at least one Xist cloud for each of the knockdowns. For additional images of multicellular colonies see Figure 7—figure supplement 2A. (D) Bar plot summarizing the percentage of Xist clouds for individual knockdowns in differentiating (DAY3) female ESCs for individual knockdowns. Cells were divided into three categories: cells carrying no Xist clouds (white), single Xist cloud (light green), or two Xist clouds (dark green). For quantifications, see Figure 7—figure supplement 2B. (E) RNA-FISH for Xist (green) in: scrambled control, shMsl1-, shMsl2-, and shMof-treated differentiating (DAY3) female ESCs. Here, we show examples of individual nuclei carrying different patterns of Xist accumulation. Percentages correspond to the frequency of the shown Xist pattern within the population of cells. White arrows denote Xist foci; dashed lines indicate nuclei borders. For quantifications see Figure 7—figure supplement 2B.

DOI: http://dx.doi.org/10.7554/eLife.02024.022

Figure 7—figure supplement 1. Depletion of MSL1 and MSL2 leads to occasional accumulation and spreading of Xist in undifferentiated ESCs.

(A) RNA-FISH for Huwe1 (red) and Xist (green) in shScrambled- (top left) and shMof- (top right), shMsl1- (bottom left) and shMsl2-treated (bottom right) female ESCs. Shown here are additional examples of RNA-FISH for multicellular colonies and individual cells exhibiting Xist-mediated coating (Figure 7A). White boxes indicate cells enlarged in Figure 7A. White arrows denote Huwe1 and Xist foci. Dashed lines indicate nuclei borders. For all experiments, nuclei were counterstained with DAPI (blue). (B) Summary of RNA-FISH for Xist and Huwe1. The number of green dots indicates the number of X chromosomes within the cell while the larger dot indicates Xist accumulation. Cells were classified into three phenotypic groups with cells showing sharp, localized Xist signals (once or twice) or Xist ‘clouds’. The percentages indicate how many cells per population showed the respective phenotype. (C) Corresponding to Figure 7A. Summary of the total cell counts from Xist and Huwe1 RNA-FISH in indicated knockdowns.

DOI: http://dx.doi.org/10.7554/eLife.02024.023

Figure 7—figure supplement 2. Depletion of MSL1 and MSL2 lead to enhanced Xist accumulation in differentiating ESCs.

(A) RNA-FISH for Huwe1 (red) and Xist (green) in shScrambled-, shMsl1-, and shMsl2-treated differentiating (DAY3) female ESCs. Shown here are additional examples of RNA-FISH for multicellular colonies (Figure 7C). Dashed lines indicate nuclei borders. For all experiments, nuclei were counterstained with DAPI (blue). (B) Corresponding to Figure 7C–E. Summary of the total cell counts from Xist RNA-FISH in indicated knockdowns. Percentage of cells with respective phenotype indicated in red.

DOI: http://dx.doi.org/10.7554/eLife.02024.024