Haploid mouse germ cell precursors from embryonic stem cells reveal Xist activation from a single X chromosome.

Eishi Aizawa,Corinne Kaufmann,Remo Freimann,Sarah Boigner,Anton Wutz,Giulio Di Minin,Sarah Sting

doi:10.1016/j.stemcr.2021.11.006

Abstract

SummaryMammalian haploid cells have applications for genetic screening and substituting gametic genomes. Here, we characterize a culture system for obtaining haploid primordial germ cell-like cells (PGCLCs) from haploid mouse embryonic stem cells (ESCs). We find that haploid cells show predisposition for PGCLCs, whereas a large fraction of somatic cells becomes diploid. Characterization of the differentiating haploid ESCs (haESCs) reveals that Xist is activated from and colocalizes with the single X chromosome. This observation suggests that X chromosome inactivation (XCI) is initiated in haploid cells consistent with a model where autosomal blocking factors set a threshold for X-linked activators. We further find that Xist expression is lost at later timepoints in differentiation, which likely reflects the loss of X-linked activators. In vitro differentiation of haploid PGCLCs can be a useful approach for future studies of potential X-linked activators of Xist.

Highlights

In mice, the germline is specified from proximal epiblast, and primordial germ cells (PGCs) are segregated from somatic lineages in the embryo
Differentiation of haploid ESCs (haESCs) into haploid primordial germ cell-like cells (PGCLCs) To investigate the feasibility of germ cell differentiation from haESCs we followed a previously established protocol (Hayashi et al, 2011)
PGCLCs appeared between days 6 and 8 and were identified by coexpression of the SSEA1 and integrin b3 surface markers (Figures 1C and 1D)

Summary

Introduction

The germline is specified from proximal epiblast, and primordial germ cells (PGCs) are segregated from somatic lineages in the embryo. PGC differentiation has been recapitulated in cultures of mouse embryonic stem cells (ESCs), which enabled the generation of functional gametes (Hayashi et al, 2011, 2012). Mammalian dosage compensation is facilitated by inactivation of one of the two X chromosomes in female cells (Lyon, 1961). X chromosome inactivation (XCI) is initiated by the long noncoding Xist RNA, which is expressed from, and accumulates over, the inactive X chromosome (Xi) before X-linked gene repression (Galupa and Heard, 2018). In the developing female epiblast, two active X chromosomes (Xas) are present before embryonic day (E) 5.5, when random XCI is initiated (Mak et al, 2004). Thereafter, the Xi is maintained in somatic lineages, but Xi reactivation is observed in the female germline (Sugimoto and Abe, 2007)

Results

Discussion

Conclusion