Xist-dependent imprinted X inactivation and the early developmental consequences of its failure

Abstract

The long noncoding RNA Xist is expressed from only the paternal X chromosome in mouse preimplantation female embryos and mediates transcriptional silencing of that chromosome. In females, absence of Xist leads to postimplantation lethality. Here, through single-cell RNA sequencing of early preimplantation mouse embryos, we found that the initiation of imprinted X-chromosome inactivation absolutely requires Xist. Lack of paternal Xist leads to genome-wide transcriptional misregulation in the early blastocyst and to failure to activate the extraembryonic pathway that is essential for postimplantation development. We also demonstrate that the expression dynamics of X-linked genes depends on the strain and parent of origin as well as on the location along the X chromosome, particularly at the first 'entry' sites of Xist. This study demonstrates that dosage-compensation failure has an effect as early as the blastocyst stage and reveals genetic and epigenetic contributions to orchestrating transcriptional silencing of the X chromosome during early embryogenesis.

Figure 1. Single cell RNA sequencing of early hybrid embryos and dosage compensation mechanisms.

(a) Schematic illustration of the single cell experiment and the harvested stages during pre-implantation mouse development. Time windows showing the persistence of maternal mRNA pool, activation of zygotic gene expression and Xp inactivation are indicated. (b) Principal component analysis (PCA) of single oocytes and pre-implantation blastomeres (2C to blastocysts) based on scRNA data. Different stages are designed by different colors. n= 6 to 30 cells per stage (details of each single cell are in Supplementary Data Set 1). (c) Differences in ratio of X-chromosome expression levels by autosomal expression levels, between 2-cell stage to blastocyst, using Dunn¹s test (Kruskal-Wallis), p<0.001 to **. Boxplots represent median with lower and upper quartiles. (d) Allele-specific expression ratios for genes on autosomes (plain red, BC or yellow CB) and on X chromosomes (dashed red, BC or yellow, CB) in female single blastomeres (2-cell to blastocyst) from BC and CB crosses. Allele-specific proportion represents the number of reads mapped to the paternal genome divided by the total number of paternal and maternal reads mapped for each gene. Boxplots represent medians with lower and upper quartiles. e) Examples of scRNA expression dynamics of three X-linked genes with their classification as “early inactivated”, “intermediate inactivated” or “escapee” (as used in Patrat et al, 2009 14) (see also Supplementary Figure 2). Mean percentage of parental origin transcripts is represented between oocytes and blastocyst.

Figure 2. Kinetics of silencing of X-linked genes over the entire X chromosome during imprinted XCI in different strains.

The mean allele-specific expression ratios per embryonic stage for each informative and expressed X-linked gene in 4-cell to blastocyst stage female embryos are represented as heatmaps, with strictly maternal expression (ratio ≤0.15) in red and strictly paternal expression (ratio ≥0.85) in blue. Color gradients are used in between these two values as shown in the key. Genes are ordered by genomic position (centromere top, telomere bottom). Data from CB (left) and BC (right) female embryos are shown (for thresholds see Online Method) and arrows highlight examples of early silenced or escapee genes. n= 125 informative X-linked genes in common for CB and BC crosses.

Figure 3. Different genes show different kinetics of silencing associated with their chromosomal position and Xist “entry” site localization.

(a) X-linked genes are clustered based on their silencing kinetics as “early” (silenced at 16-cell or earlier), “intermediate” (silenced at 32-cell), “late” (silenced at blastocyst), “biased” (maternally biased) and “escapee” (Esc, not silenced). The allelic ratio of each gene represents the number of reads mapped on the paternal genome divided by the total number of reads mapped and is represented at 4-cell, 16-cell, 32-cell and blastocyst stages from single female blastomeres. Further information is provided in Supplementary Table 1 and Online Methods. n= 137 X-linked genes (89 with consistent silencing kinetics between BC and CB crosses and 48 BC or CB-specific). (b) Parental expression ratios of X-linked genes in female blastocysts in BC and CB strains. Each dot represents a single gene. The upper and lower sections represent data respectively from BC and CB embryos. Xist is represented by a red dot. Green and orange dots represent genes that escape from early XCI respectively in both strains or in strain-specific manner. Further information on escapees is found in Supplementary Table 2. n= 125 common X-linked genes. (c) Box plot representing the distribution of the genomic distances to Xist locus (in Mb) for the different clusters of genes. “Transcription Start Site (TSS) of each gene has been used to measure the distance to Xist. p<0.05 corresponds to * by Dunn’s test.” (d) Allelic expression of X-linked genes classified by their relative position to Xist “entry” sites (as identified during XCI induction in ESCs20): “inside” (TSS located in a Xist “entry” site), “next to” (TSS located less than 100 kb to an “entry” site) and “outside” (over 100 kb from an “entry” site). By Dunn’s test; p<0.05 corresponds to *. Consistent or strain-specific genes have been used. Boxplot represent median with lower and upper quartiles.

Figure 4. Paternal knockout of Xist impaired XCI, dosage compensation and differentiation pathways.

(a) Differences in ratio of X-chromosome expression levels by autosomal expression levels, between 8-cell stage to blastocyst in CB females (left panel) and Xist^pat∆ CB females (with a paternally inherited knock-out allele) (right panel). Boxplots represent median with lower and upper quartiles. (b) Heatmap representing allele-specific mean expression from 8-cell to blastocyst stage of X-linked genes (as in Figure 2) in Xist^pat∆ mutant single cells. Strictly maternally expressed genes (allelic ratio ≤0.15) are represented in red and strictly paternally expressed genes (allelic ratio ≥0.85) in blue. Color gradients are used in between and genes have been ordered by genomic position. Tsix was included in the heatmap if it was expressed in at least 2 single cells per stage, even though it did not reach the expression threshold used (RPRT>4 and expressed in at least 25% of the cells of each stage and cross with a minimum of 2 cells). n = 122 genes. (c) Major down-regulated genes and pathways detected between CB wt and CB Xist^pat∆ females extracted from Supplementary Data Set 2, using QIAGEN’s Ingenuity Pathway Analysis (IPA) software (Supplementary Data Set 3). Color code for arrows, red: leads to inhibition; blue: leads to activation; orange: findings consistent with state of downstream molecule; grey: effect not predicted. (d) Expression data of candidate genes from wt CB (black) and Xist^pat∆ CB (red) females, extracted from scRNAseq. Mean of expression is represented in Reads Per Retro-Transcribed length per million mapped reads (RPRT) during early development (8-cell to blastocyst stages). Gapdh gene is a control housekeeping gene. n= 4 to 30 cells per stage and genotype. By Kruskal-Wallis test; p<0.05 corresponds to *.

Figure 5. Abnormal Sox17 and Rhox5 patterns in Xist^pat∆ female blastocysts.

Maximum intensity projection of wt and Xist^pat∆ E4.25 blastocysts analyzed by immunofluorescence against Sox17 (a, c) or Rhox 5 (b, d). Staining for Sox17 or Rhox5 is in red, DAPI is in grey. Scale bar represents 20µm. Percentage of positive cells have been assessed and summarized as the median + s.e.m. for Sox17 (e) and Rhox5 (f). Numbers of embryos are indicated under each genotype. By Kruskal-Wallis test; p<0.05 and p<0.001 correspond respectively to * and **. Average distribution of positive single cell fluorescence was represented by measuring the corrected total cell fluorescence using ImageJ software (Fiji, NIH) for Sox17 (g) and Rhox5 (h) and tested by Kolmogorov-Smirnov test. All cells under 10,000 and 5,000 for total cell fluorescence, respectively for Sox17 and Rhox5, have been considered as negative.