High-sensitivity in situ detection of Sox21 reveals transcript asymmetry from the zygote stage

The first lineage decision in mammalian development segregates embryonic and extra-embryonic fates, yet the timing of this specification remains under debate. While our previous lineage tracing studies in mice and human suggested that this decision is initiated as early as the 2-cell stage, prevailing models place its onset to the 16-cell stage embryo when the first inside cells form. In mice, we have found that heterogeneous expression of Sox21 at the 4-cell stage contributes to fate specification, with higher SOX21 levels biasing cells toward embryonic fates (future foetus). Here, we investigated whether this heterogeneity originates even earlier. Because conventional single-cell RNA-seq has failed to detect mRNA asymmetries prior to the 4-cell stage, we employed a more sensitive approach, confocal imaging combined with hybridization chain reaction (HCR), to track Sox21 mRNA expression from the zygote through the 4-cell stage. This single-molecule technique revealed pronounced heterogeneity in Sox21 transcript levels as early as the 2-cell stage. Intriguingly, Sox21 mRNA appears to be preferentially expressed from the male pronucleus during minor zygotic genome activation, perhaps as a consequence of H3K27me3 enrichment in the maternal pronuclei and given that haploid parthenogenetic embryos lack SOX21 protein at the 4-cell stage. These findings provide evidence that Sox21 mRNA heterogeneity arises earlier than previously recognized, namely at the 2-cell stage. Our work supports the emerging view that spatial mRNA distribution, long appreciated in non-mammalian embryos, might also contribute to lineage specification in mammals.