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

In Mammals, the difference in sex-chromosome constitution between males (XY) and females (XX) has led to the evolution of dosage compensation strategies, including silencing of an entire X chromosome in females. In mice, X chromosome inactivation (XCI) first occurs in the pre-implantation embryo. Here the non-coding Xist RNA is expressed only from the paternal allele and the paternal X (Xp) becomes inactivated. The Xp is reactivated in the inner cell mass and this is followed by random XCI in the embryo proper, while in extra-embryonic tissues the Xp remains inactive. Although random XCI initiation is thought to be fully Xist-dependent in the mouse, initiation of imprinted XCI was reported to be Xist-independent. Furthermore, the chromosome-wide dynamics of XCI in early embryos of reciprocal inter-specific crosses, which better reflect a natural situation, have never been investigated. Here we report that the expression dynamics of X-linked genes depends both on strain and parent of origin, as well as on X chromosome location, using single-cell RNA-sequencing (scRNAseq) of early mouse embryos. We also demonstrate that initiation of imprinted XCI absolutely requires Xist and that its absence leads to genome-wide transcriptional misregulation in the early blastocyst, with massive over-expression of specific genes such as Rhox5 and failure to activate the extra-embryonic pathway essential for early post-implantation development. This study provides important insights into the transcriptional and allelic dynamics of the X chromosome and the first evidence of dosage compensation failure as early as the blastocyst stage. Overall design: 182 single-cells RNAseq from oocytes and hybred males and females pre-implantation embryos, with or without deletion of the paternal Xist allele.

在哺乳动物中，雄性（XY）与雌性（XX）的性染色体组成差异驱动了剂量补偿策略的演化，其中包含雌性整条X染色体的沉默过程。在小鼠中，X染色体失活（X chromosome inactivation, XCI）最早发生于植入前胚胎阶段：此时非编码RNA Xist仅从父本等位基因表达，父本X染色体（Xp）随即发生失活。随后，Xp在内细胞团中被重新激活，胚胎本体随即启动随机XCI；而在胚外组织中，Xp始终保持失活状态。尽管学界普遍认为小鼠中随机XCI的起始完全依赖Xist，但已有研究报道印记式XCI的起始不依赖Xist。此外，能更真实反映自然状态的互交种间杂交小鼠早期胚胎中，XCI的全染色体动态变化尚未被系统探究。本研究通过对小鼠早期胚胎开展单细胞RNA测序（scRNAseq），发现X连锁基因的表达动态同时受品系、亲本起源以及X染色体上的位置影响。本研究同时证实，印记式XCI的起始绝对依赖Xist；Xist的缺失会导致早期囊胚出现全基因组转录失调，具体表现为Rhox5等特定基因的大量过表达，且无法激活早期植入后发育所必需的胚外通路。本研究为X染色体的转录与等位基因动态变化提供了重要见解，并首次提供了早在囊胚阶段即出现剂量补偿失败的实验证据。整体实验设计：从卵母细胞以及带有或缺失父本Xist等位基因的杂交雌雄小鼠植入前胚胎中，共获取182个单细胞进行RNA测序。