Single-cell analyses of X Chromosome inactivation dynamics and pluripotency during differentiation

Abstract

Pluripotency, differentiation, and X Chromosome inactivation (XCI) are key aspects of embryonic development. However, the underlying relationship and mechanisms among these processes remain unclear. Here, we systematically dissected these features along developmental progression using mouse embryonic stem cells (mESCs) and single-cell RNA sequencing with allelic resolution. We found that mESCs grown in a ground state 2i condition displayed transcriptomic profiles diffused from preimplantation mouse embryonic cells, whereas EpiStem cells closely resembled the post-implantation epiblast. Sex-related gene expression varied greatly across distinct developmental states. We also identified novel markers that were highly enriched in each developmental state. Moreover, we revealed that several novel pathways, including PluriNetWork and Focal Adhesion, were responsible for the delayed progression of female EpiStem cells. Importantly, we "digitalized" XCI progression using allelic expression of active and inactive X Chromosomes and surprisingly found that XCI states exhibited profound variability in each developmental state, including the 2i condition. XCI progression was not tightly synchronized with loss of pluripotency and increase of differentiation at the single-cell level, although these processes were globally correlated. In addition, highly expressed genes, including core pluripotency factors, were in general biallelically expressed. Taken together, our study sheds light on the dynamics of XCI progression and the asynchronicity between pluripotency, differentiation, and XCI.

Sex Differences in Early Embryogenesis: Inter‐Chromosomal Regulation Sets the Stage for Sex‐Biased Gene Networks

“…We confirmed this in a meta-analysis of single cell RNAsequencing studies, [52] which raises questions on the mechanisms of equalization and on what happens to these genes after the cells undergo X chromosome inactivation. We confirmed this in a meta-analysis of single cell RNAsequencing studies, [52] which raises questions on the mechanisms of equalization and on what happens to these genes after the cells undergo X chromosome inactivation.…”

Mammalian embryo comparison identifies novel pluripotency genes associated with the naïve or primed state

“…These intersections also highlight that some mEpiSC-associated genes are already upregulated in serum-mESCs (29 out of the 44 genes upregulated between 2i-mESCs and mEpiSCs are also upregulated between 2i- and serum-mESCs, ) while various 2i-mESC-associated genes are significantly decreased in serum-mESCs (10 out of the 16 genes downregulated between 2i-mESCs and mEpiSCs are already downregulated between 2i- and serum-mESCs, ). As such, serum-mESCs can be considered as a transitional stage between naïve and primed pluripotency, which is in keeping with what has recently been reported (Chen et al, 2016).

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Landscape of X chromosome inactivation across human tissues

“…1), as demonstrated recently in mouse studies 19-22 , and in human fibroblasts 23 and preimplantation development 24 . To directly profile XCI in human samples, we examined scRNA-seq data in combination with deep genotype sequences from 940 immune-related cells from four females: 198 cells from LCLs sampled from three females of African (Yoruba) ancestry, and 742 blood dendritic cells from a female of Asian ancestry 25 (Fig.…”