Heterogeneity in Oct4 and Sox2 Targets Biases Cell Fate in 4-Cell Mouse Embryos

Mubeen Goolam,Thierry Voet,Antonio Scialdone,John C Marioni,Anna Hupalowska,Iain C Macaulay,Magdalena Zernicka-Goetz,Agnieszka Jedrusik,Sarah J L Graham

doi:10.1097/01.ogx.0000488738.30718.bf

Abstract

The major and essential objective of pre-implantation development is to establish embryonic and extra-embryonic cell fates. To address when and how this fundamental process is initiated in mammals, we characterize transcriptomes of all individual cells throughout mouse pre-implantation development. This identifies targets of master pluripotency regulators Oct4 and Sox2 as being highly heterogeneously expressed between blastomeres of the 4-cell embryo, with Sox21 showing one of the most heterogeneous expression profiles. Live-cell tracking demonstrates that cells with decreased Sox21 yield more extra-embryonic than pluripotent progeny. Consistently, decreasing Sox21 results in premature upregulation of the differentiation regulator Cdx2, suggesting that Sox21 helps safeguard pluripotency. Furthermore, Sox21 is elevated following increased expression of the histone H3R26-methylase CARM1 and is lowered following CARM1 inhibition, indicating the importance of epigenetic regulation. Therefore, our results indicate that heterogeneous gene expression, as early as the 4-cell stage, initiates cell-fate decisions by modulating the balance of pluripotency and differentiation.

Highlights

When in mammalian development cells first start to differ from each other and whether these first differences play any role in cell-fate specification remain key open questions
To address when and how this fundamental process is initiated in mammals, we characterize transcriptomes of all individual cells throughout mouse pre-implantation development
To assess the quality of the data we compared, across cells in each batch, three metrics: the fraction of reads mapped to endogenous RNA molecules, the number of genes with more than 10 reads per million, and the fraction of reads mapped to mitochondrial genes (Stegle et al, 2015)

Summary

Introduction

When in mammalian development cells first start to differ from each other and whether these first differences play any role in cell-fate specification remain key open questions. The embryonic lineage is pluripotent and will give rise to the fetus, while the extra-embryonic lineages will differentiate into supportive structures critical for embryo implantation and fetal development, the placenta, and yolk sac (Takaoka and Hamada, 2012; Zernicka-Goetz et al, 2009) How and when these lineages start to separate in morphologically homogenous cells has been very difficult to dissect in mammals. More recent evidence has suggested that cells as early as the 4-cell stage become heterogeneous, exhibiting differences in developmental fate and potential (Bischoff et al, 2008; Piotrowska-Nitsche et al, 2005; Tabansky et al, 2013) and in the activity of specific cell-fate regulators (Burton et al, 2013; Plachta et al, 2011; Torres-Padilla et al, 2007) This heterogeneity indicates the possibility that the breaking of embryo symmetry starts earlier than expected, prior to differences in cell position and polarity evident from the 16-cell-stage onward (Fleming, 1987; Johnson and Ziomek, 1981). Finding causal links between this early heterogeneity and later lineage divergence has proved extremely difficult because the key evidence—differences in gene expression patterns between individual cells that regulate cell fate—has, until now, been hard to identify due to technical limitations

Results

Discussion

Conclusion