Heterogeneities in Nanog Expression Drive Stable Commitment to Pluripotency in the Mouse Blastocyst

Abstract

Summary The pluripotent epiblast (EPI) is the founder tissue of almost all somatic cells. EPI and primitive endoderm (PrE) progenitors arise from the inner cell mass (ICM) of the blastocyst stage embryo. The EPI lineage is distinctly identified by its expression of pluripotency-associated factors. Many of these factors have been reported to exhibit dynamic fluctuations of expression in embryonic stem cell cultures. Whether these fluctuations correlating with ICM fate choice occur in vivo remains an open question. Using single-cell resolution quantitative imaging of a Nanog transcriptional reporter, we noted an irreversible commitment to EPI/PrE lineages in vivo. A period of apoptosis occurred concomitantly with ICM cell fate choice, followed by a burst of EPI-specific cell proliferation. Transitions were occasionally observed from PrE-to-EPI, but not vice versa, suggesting that they might be regulated and not stochastic. We propose that the rapid timescale of early embryonic development prevents frequent fluctuations in cell fate.

Lineage specification in the mouse preimplantation embryo

“…This was previously suggested by cell lineage tracing experiments (Chazaud et al, 2006;Meilhac et al, 2009) and confirmed by live cell tracking with a Nanog-GFP reporter (Xenopoulos et al, 2015). Interestingly, however, it was shown (Xenopoulos et al, 2015) that a few cells expressing low levels of Nanog-GFP until a relatively late blastocyst stage can strongly upregulate Nanog-GFP expression eventually, which is suggestive of fate reversion. The relative levels of NANOG and GATA6 were not examined in this study and it might be the balance between the two transcription factors that is important rather than their absolute amounts.…”

“…This gives rise to a mutually exclusive 'salt and pepper' expression pattern of NANOG and GATA6 by embryonic day (E) 3.75. Cell lineage tracing and live cell tracking analyses (Chazaud et al, 2006;Kurimoto et al, 2006;Plusa et al, 2008;Meilhac et al, 2009;Xenopoulos et al, 2015) show that this process occurs in individual ICM cells asynchronously, spanning from ∼E3.0-E3.75 (Gerbe et al, 2008;Plusa et al, 2008;Bessonnard et al, 2014). Therefore, a few cells coexpressing both transcription factors can still be identified at ∼E3.75 in some embryos, and the emergence of NANOG + / GATA6 − EPI cells is currently the first known sign of the specification process.…”

“…None of these hypotheses has yet been discarded or favoured; however, mathematical simulations mimicking high transcriptional noise of all the core factors indicate that this internal noise is unlikely to be the initiating mechanism (Bessonnard et al, 2014;DeMot et al, 2016). Thus, although the initial step is known to be the commitment of one or a few ICM cells to an EPI fate at ∼E3.0, always preceding PE specification in other cells (Bessonnard et al, 2014;Xenopoulos et al, 2015), we still do not know why FGF4 and/or NANOG expression increases in just a few ICM cells.…”

“…It is possible that individual embryos are unique in the degree, pattern and variety of heterogeneity that they exhibit. Directly monitoring this heterogeneity in live embryos (Plusa et al, 2005;Dietrich et al, 2015;Xenopoulos et al, 2015) and the development of more sophisticated gene manipulation strategies will be important for gaining a full understanding of the functional consequences of heterogeneity in early blastomeres.…”

Venus trap in the mouse embryo reveals distinct molecular dynamics underlying specification of first embryonic lineages

“…Given the dynamicity and stochastic molecular heterogeneity, mechanistic understanding of the first lineage specification requires quantitative description of the gene expression dynamics in living embryos during morphogenesis at single-cell resolution [16]. However, to date, only a limited number of fluorescence gene expression reporter mice suitable for live imaging are available [5,[17][18][19], quantitative analysis of gene expression dynamics has begun only recently [19], and it has not been systematically integrated with cell lineage segregation pattern.…”