Transcriptome of mouse preimplantation development [Rlim KO]

Purpose: The goals of this study are to establish a roadmap of XCI and compare the transcriptomes of WT and Rlim KO embryos during X chromosome inactivation. Methods: mRNA profiles of 175 preimplantation embryos WT and KO for Rlim were elucidated by RNA-seq at various stages. Trophoblasts isolated from blastocyst outgrowths were also included. The sequence reads that samples where gender could be determined and that passed quality filters were analyzed at the level of autosomes, X xhromosomes as well as single genes. Results: Using single cell RNA-seq technology on 175 whole preimplantation embryos, we obtained about 2.95 million sequence reads per sample. Reads were normalized to autosomal gene expression. Gender of each embryo was determined by expression of Y-linked genes and Xist. Data analysis showed normal expression profiles of marker genes for epiblast and trophoblast cell types during preimplantation development. Comparing Xist expression profiles in embryos WT and KO shows that Rlim is not required for initiation of Xist transcription but for upregulation of Xist expression. Moreover, our results identify two waves of XCI during preimplantation development: One that occurs at Morula stages that is Rlim-independent and one at blastocyst stages that in dependent on Rlim. Conclusions: Our study represents the first detailed mouse preimplantation transcriptome. Our results show that Rlim is required for a second wave of imprinted XCI that occurs in female embryos at blastocyst stages. Global mRNA profiles of whole preimplantation embryos were generated by deep sequencing.研究目的：本研究旨在确立X染色体失活（X-chromosome inactivation，XCI）的研究路线图，并比较X染色体失活过程中野生型（Wild Type，WT）与Rlim敲除（Knockout，KO）胚胎的转录组。 研究方法：针对175个Rlim野生型与敲除型的植入前胚胎，在不同发育阶段通过RNA测序（RNA-seq）解析其mRNA表达谱；同时纳入从囊胚培养物中分离得到的滋养层细胞样本。对可确定性别且通过质量质控过滤的样本的测序读段，分别在常染色体、X染色体以及单基因水平开展数据分析。 研究结果：本研究对175枚完整植入前胚胎采用单细胞RNA测序技术，每个样本平均获得约295万条测序读段。读段以常染色体基因表达量作为参照进行归一化处理。通过Y连锁基因与Xist的表达水平鉴定每枚胚胎的性别。数据分析显示，植入前发育过程中，上胚层与滋养层细胞类型的标志物基因表达谱均处于正常水平。对比野生型与敲除型胚胎的Xist表达谱发现，Rlim并非Xist转录起始所必需，而是参与Xist表达的上调过程。此外，本研究明确了植入前发育过程中X染色体失活的两个阶段：一类发生于桑椹胚阶段，不依赖Rlim；另一类发生于囊胚阶段，依赖Rlim。 研究结论：本研究构建了首个详尽的小鼠植入前胚胎转录组数据集。研究结果表明，Rlim是雌性胚胎囊胚阶段发生的第二波印记型X染色体失活所必需的调控因子。本研究通过深度测序获得了完整植入前胚胎的全局mRNA表达谱。